CN109843931A - Immunomodulatory compounds - Google Patents

Abstract

Disclose the compound for adjusting immune response.More specifically, the invention discloses the PD-L2 of the oligomerization form for adjusting Th1 immune response.The compound of the present invention can be used for a series of disease that Th1 are mediated, including pathogenic infection and excess proliferative disease.

Description

Immunomodulatory compounds

Technical field

The present invention relates generally to the compounds for adjusting immune response.More particularly, the present invention relate to adjust Th1 The oligomerization form of the PD-L2 of immune response.The compounds of this invention can be used for a series of disease that Th1 are mediated, including cause of disease sexuality Dye and excess proliferative disease.

Background technique

Apoptosis albumen 1 (PD-1) is considered as the important participant in immunological regulation, by it as effect The braking of T cell is answered, and reduces the immune response in tissue microenvironment.PD-1 is expressed in the T cell of activation, including immune suppression Property CD4 processed⁺CD8 T cell (Treg) and exhausted⁺T cell, also in B cell, myeloid dendritic cell (MDC), monocyte, thymus gland It is expressed on cell and natural kill (NK) cell.This extensive PD-1 expression prompt is effectively immune and maintains T cell stable state institute Need PD-1 signal transduction path broad sense (Gianchecchi et al., Autoimmun.Rev.12:1091-100, 2013)。

It is worth noting that, PD-1 signal transduction path helps to maintain the maincenter and peripheral tolerance of normal individual.In chest In gland, the interaction of PD-1 and its ligand inhibits positive selection, to inhibit CD4^-CD8^-Double negative cells are to CD4⁺CD8⁺ The conversion (Keir et al., J.Immunol.175:7329-7379,2005) of double positive T cells.PD-1 signal transduction is also responsible for pressing down Autoreactivity processed and the inflammatory effector T cell for escaping Solid phase, to avoid subsidiary immune-mediated tissue damage (Keir Et al., J.Exp.Med.203:883-895,2006).

There are two types of known ligands for PD-1 tool: 1 (PD-L1 of albumen death ligand；Freeman et al., J.Exp.Med.192: 1027-34,2000), also referred to as B7-H1 (Dong et al., Nat.Med.5:1365-9,1999) and albumen are dead in the mankind Die 2 (PD-L2 of ligand；Latchman et al., Nat.Immunol.2:261-8,2001), also referred to as B7-DC (Tseng et al., J.Exp.Med.193:839-46,2001).The expression pattern of PD-L1 and PD-L2 is very different.PD-L1 is by panimmunity cell Constitutive expression is carried out with nonimmune cell, and is seemed in most of normal tissue cells in the presence of strong inflammatory signals Up-regulation (Matzinger et al., Nat.Rev.Immunol.11:221-230,2011；M ü hlbauer et al., J.Hepatol.45:520-528,2006；Pinchuk et al., Gastroenterol.135:1228-1237,2008； Stanciu et al., J.Infect.Dis., 193:404-412,2006).In contrast, the composing type basal expression of PD-L2 with PD-L1 is compared to lower, although PD-L2 expression is initially considered as limited to antigen presenting cell, as monocyte, macrophage and Dendritic cells (DC) (Latchman et al., Nature Immunol.2:261-268,2001；Yamazaki et al., J.Immunol.169:5538-5545,2002), have recently several groups research shows that in various other immunocytes and non-can exempt from The expression of PD-L2 is induced on epidemic disease cell, this, which depends on microenvironment, stimulates (Kinter et al., J.Immunol.181:6738- 6746,2008；Zhong et al., Eur.J.Immunol.37:2405-2410,2007；Messal et al., Mol.Immunol.48:2214-2219,2011；Lesterhuis et al., Mol.Immunol.49:1-3,2011).

The microenvironment cell unconventionality expression PD-1 and its ligand of malignant cell and surrounding.In tumor microenvironment, PD-1 exists Height is expressed in most of tumor infiltrating lymphocyte (TIL) from many different tumor types, and local effect is inhibited to exempt from Epidemic disease response.The TIL expression of PD-1 and effector function impaired (cell factor generates and to the cytotoxic effects of tumour cell) and/ Or the bad result of several tumor types it is related (Thompson et al., Clin.Cancer Res.13:1757-1761,2007； Zhang et al., Mol.Immunol.7:389-395,2010；114:1537-1544 Ahmadzadeh M et al., Blood, 2009；Shi et al., Int.J.Cancer 128:887-896,2011), including clear-cell carcinoma, metastasis melanin tumor and Gastric cancer, breast cancer, oophoroma, cancer of pancreas and lung cancer.Similarly, it has been observed that PD-L2 is raised in the subset of human tumour, And (Rozali et al., Clin.Dev.Immunol.2012:656340,2012) related to bad result sometimes.

In view of its latent effect in tumor microenvironment in immunosupress relevant to cancer, it has been suggested that will target PD-1/PD- ligand pathways are as attractive therapeutic strategy.Some researchs in this respect have studied blocking antibody To the therapeutic effect of PD-1/PD-L1 approach, it was demonstrated that tumor control rate (Curran et al., the Proc.Natl.Acad of enhancing Sci.USA 107:4275-4280,2010；Iwai et al., Proc.Natl.Acad Sci.USA 99:12293-12297, 2002；Pilon-Thomas et al., J.Immunol.184:3442-3449,2010；Zhang et al., Blood 114:1545- 1552).However, few research block PD-L2 as specific therapeutic strategy.Although being used in seldom research PD-L2 blocking strategy, but this always with targeting PD-L1 combine (Parekh et al., J.Immunol.182:2816-1826, 2009；He et al., J.Immunol.173:4919-4928,2004), it can not infer the true value of anti-PD-L2 strategy.

Summary of the invention

The present invention is based partially on such a determination, i.e. PD-L2 with antigen specific immune effector cell (IEC) (institute Stating immune effector cell includes T lymphocyte) interaction cell (such as antigen presenting cell (APC)) on expression and Th1 The severity of related disease is negatively correlated and PD-L2 is to establish the immune necessary condition of Th1.It has also been found that PD-L2 assembles It can inhibit the combination of PD-L1 and PD-1 on the surface of this IEC- interaction cell, to inhibit PD-L1 to IEC's Immune suppression function., it is surprising that the present inventor further defines, compared to the combination of dimer PD-L2 and PD-1, oligomerization Degree has significant higher affinity greater than 2 PD-L2 oligomer, and this " the PD-L2 oligomer of higher amount grade " can be with PD-L1 is significantly reduced to the inhibiting effect of IEC function, including CD4⁺T cell function.As described below, these discoveries have been practiced To for adjusting in Th1 immune novel agent and method.

Therefore, in one aspect, the present invention provides can be used for stimulating or enhance the immune polypeptide complex of Th1, this is multiple Object is closed usually by shown in formula (I):

[P]_n (I)

Wherein:

P, independently represents protein molecular when occurring every time, the protein molecular includes PD-L2 polypeptide, by PD-L2 polypeptide Composition is substantially made of PD-L2 polypeptide；And

N represents the integer greater than 2.

Suitably, P lacks tumour or the relevant new vessels targeting structural domain of tumour.

In some embodiments, PD-L2 polypeptide include the soluble fraction of PD-L2, be made of the soluble fraction of PD-L2, Or be substantially made of the soluble fraction of PD-L2, illustrated examples include the PD-L2 extracellular domain or no signal peptide of signal peptide PD-L2 extracellular domain.In specific embodiments, protein molecular lacks PD-L2 transmembrane domain and PD-L2 cytoplasmic domains One or both of.

Suitably, n be >=3, >=4, >=5, >=6, >=7 or >=8.In the illustrated examples of this type, n be≤100 ,≤ ≤ 30 or≤20 50,.In specific embodiments, n is 3 to 20, is suitably 4 to 16, is more suitably 8 to 12.

Protein molecular chemical coupling is together to form polypeptide complex.Or single protein molecular also includes at least one Oligomerization domain, the oligomerization domain promote the self assembly of protein molecular to form polypeptide complex.In these implementations In scheme, typically, at least one oligomerization domain and PD-L2 polypeptide are operably connected to form single-chain chimeric polypeptide. In the embodiment that at least one oligomerization domain is operatively connected to PD-L2 polypeptide, on the other hand the present invention provides A kind of protein molecular, it includes the above and broadly described PD-L2 polypeptide of elsewhere herein, by above and elsewhere herein Broadly described PD-L2 polypeptide composition is substantially made of the broadly described PD-L2 polypeptide of above and elsewhere herein, The PD-L2 polypeptide is operably coupled to a few oligomerization domain to form the polypeptide complex according to formula (I), wherein The oligomerization domain promotes the self assembly of the protein molecular.Oligomerization domain is operably coupled to the PD-L2 The upstream (i.e. amino terminal) of polypeptide and/or downstream (i.e. carboxyl terminal).For example, can be operated at least one oligomerization domain Ground is connected in the embodiment in PD-L2 polypeptide downstream, and protein molecular includes single polypeptide chain shown in formula (II), by formula (II) institute Show single polypeptide chain composition or substantially the single polypeptide chain shown in formula (II) form:

PD-L2-L-OMD_A (II)

Wherein:

PD-L2 represents PD-L2 polypeptide；

OMD_AIt is oligomerization domain, forms i subunit OMD_AOligomer (OMD_A)_i, wherein i >=3, suitably for 3,4,5 or 6；And

L is key or peptide linker.

Alternatively, protein molecular include single polypeptide chain, the single polypeptide chain shown in formula (III) shown in formula (III) form or Substantially the single polypeptide chain shown in formula (III) forms:

PD-L2-L-OMD_A-L-OMD_B (III)

Wherein:

OMD_AIt is oligomerization domain, forms i subunit OMD_AOligomer (OMD_A)_i, wherein i >=2, suitably for 2,3,4,5 or 6；

L independently represents key or peptide linker when occurring every time；And

OMD_BIt is oligomerization domain, forms j subunit OMD_BOligomer (OMD_B)_j, wherein j is greater than the whole of i Number, is suitably i+1, i+2, i+3, i+4, i+5 or i+6；

In such illustrated examples, i is that 2 and j is 4 or 6.

In some embodiments, at least one oligomerization domain is operably coupled to PD-L2 polypeptide upstream, albumen Molecule forms or comprising single polypeptide chain, the single polypeptide chain shown in formula (IV) shown in formula (IV) substantially by shown in formula (IV) Single polypeptide chain composition:

OMD_A-L-PD-L2 (IV)

Wherein:

OMD_AIt is oligomerization domain, forms i subunit OMD_AOligomer (OMD_A)_i, wherein i >=3, suitably for 3,4,5 or 6；

L is key or peptide linker；And

PD-L2 represents PD-L2 polypeptide.

Alternatively, protein molecular includes that single polypeptide chain, the single polypeptide chain shown in formula (V) shown in formula (V) forms or substantially On the single polypeptide chain shown in formula (V) form:

OMD_B-L-OMD_A-L-PD-L2 (V)

Wherein:

OMD_BIt is oligomerization domain, forms j subunit OMD_BOligomer (OMD_B)_j, wherein j >=2, suitably for 2,3,4,5 or 6；

L independently represents key or peptide linker when occurring every time；And

OMD_AIt is oligomerization domain, forms i subunit OMD_AOligomer (OMD_A)_i, wherein i is greater than the whole of j Number, is suitably j+1, j+2, j+3, j+4, j+5 or j+6；And

PD-L2 represents PD-L2 polypeptide.

In such illustrated examples, j is that 2 and i is 4 or 6.

In some embodiments, individual oligomerization domain is (for example, OMD_AOr OMD_B) in binding partners, there are the following groups Dress up different oligomer.Oligomerization domain and the binding partners can be the member of specific binding pair, illustrative examples Including biotin-avidin, biotin-Streptavidin, Ag-Ab, haptens-antihapten, ligand-receptor And receptor-co-receptor.

The present invention considers to use any suitable oligomerization domain, including such as dimerization domain (for example, immune ball Albumen Fc structural domain, leucine zipper etc.), trimerising domain is (for example, Escherichia coli aspartate carbamyl-transferase (ATCase) catalytic subunit, " foldon " the trimerization sequence from T4 bacteriophage minority fibers albumen (fibritin), neck region The complementary heptapeptide repetition of peptide, people's Curosurf protein D, oligomerization coiled coil adhesin, I class enveloped virus fusion protein Area etc.), tetramerization structural domain (for example, coiled-coil domain of viscous connection albumen (tetrabrachion) of four arms), five dimerization knots Structure domain (for example, tryptophan zipper or five multimerisation domains of cartilage oligo-substrate protein (COMP) etc.) and six multimerisation domains (for example, tail portion from IgA antibody heavy chain C-terminal).

In some embodiments, at least one oligomerization domain is directly connect with PD-L2 polypeptide.In other embodiment party In case, at least one oligomerization domain is connected by peptide linker with PD-L2 polypeptide, and the peptide linker is usually by about 1 to about 100 amino acid residue (and all integer amino acid residues therebetween) compositions, normally about 1 to about 30 amino acid residue (and all integer amino acid residues therebetween), normally about 1 to about 20 amino acid residue (and all integer ammonia therebetween Base acid residue).Peptide linker may include at least one portion, and the part is selected from: promote to purify the purifying portion of the protein molecular Divide, adjust the immunological regulation part to the protein molecular immune response and assign the part of configuration flexibility.

It can be synthetically produced or protein molecular is generated by recombination method.In the embodiment that recombination generates protein molecular In, on the other hand the present invention provides a kind of nucleic acid constructs, and it includes the above and broadly described albumen of elsewhere herein The coded sequence of molecule is operably coupled to the regulating element that can be operated in host cell.

In related fields, the present invention provides a kind of host cell, contains and described extensively with elsewhere herein above Nucleic acid construct.Host cell can be protokaryon or eukaryotic host cell.

In the embodiment that protein molecular includes at least oligomerization domain, protein molecular can be under suitable conditions (for example, in aqueous solution), self assembly was to form the polypeptide complex according to formula (I).Therefore, on the other hand, the present invention provides A kind of method generating polypeptide complex, the method comprise the steps that (example under conditions of suitably forming polypeptide complex Such as, in aqueous solution), the protein molecular that above and elsewhere herein limits extensively is combined, so that it is multiple to generate polypeptide Object is closed, it includes the oligomer of n protein subunit molecule.

On the other hand, the present invention provides a kind of immune regulation composite, and it includes retouch extensively with elsewhere herein above The polypeptide complex and pharmaceutically acceptable carrier or adjuvant stated.

Polypeptide complex or composition of the invention can be used for exempting from subject or production animal moderate stimulation, initiation or enhancing Epidemic disease response, the immune response include Th1 immune response.Therefore, another aspect provides one kind in subject Stimulation, cause or enhancing immune response method, the immune response includes Th1 immune response, wherein the method includes to The subject applies the above and broadly described polypeptide complex of elsewhere herein or composition.

In related fields, the present invention provides the methods of disease relevant to Th1 or illness in treatment subject.These sides Method generally includes to apply a effective amount of above and broadly described polypeptide complex of elsewhere herein or composition to subject.

In some embodiments, when subject is accredited as immune with impaired Th1, polypeptide of the invention is answered It closes object or composition is applied to subject.The Th1 immune state of any suitable method assessment subject can be used.Advantageous Embodiment in, by the following method assess subject Th1 immune state: (1) determine be obtained from subject sample in Th1 immune state biomarker spectrum, wherein Th1 immune state biomarker spectrum includes at least one of sample Th1 The biomarker value of immune state biomarker, wherein at least one Th1 immune state biomarker include with PD-L2 and optional PD-L1 in the sample in the cell of IEC- interaction cell interaction；And (2) use institute It states biomarker value and determines indicant, wherein the indicant at least partly indicates the Th1 immune state of the subject.? In specific embodiment, IEC- interaction cell is APC, is appropriately selected from dendritic cells and macrophage.This In the representative example of type, APC is the dendritic cells for expressing CD11c.In other embodiments, IEC- interaction cell It is tumour cell.

Suitably, biomarker value at least partly indicates Th1 immune state biology mark in the sample obtained from subject Remember the concentration of object, in some embodiments of the type, biomarker object value includes Th1 immune state biomarker Abundance.In representative example, individual biomarker value includes: the expression Th1 immune state biomarker on cell surface The percentage of the IEC- interaction cell of object is (for example, PD-L2⁺Dendritic cells, PD-L2⁺Tumour cell etc.).In this seed type Some embodiments in, Th1 immune state biomarker be PD-L2 and biomarker value to be collected on IEC- mutual The measurement of PD-L2 on function cells (such as APC (such as dendritic cells) or tumour cell) surface.

When determining the Th1 immune state of subject, in some embodiments, relative to PD-L2 control level and Speech, the horizontal of PD-L2 reduces in sample, wherein the control level of the PD-L2 with that there are normal Th1 is immune or undamaged The immune correlation of Th1, it is thus determined that indicant at least partly indicates Th1 immunocompromised host.

In other embodiments, in sample PD-L2 level, the PD-L2 roughly the same with the control level of PD-L2 Control level to that there are normal Th1 immune or undamaged Th1 is immune related, it is thus determined that indicant is at least partly to refer to Show that the immune or undamaged Th1 of normal Th1 is immune.In other embodiments, relative to the control level of PD-L2, sample The horizontal of middle PD-L2 increases, the control level of the PD-L2 with there are the immune phases of normal Th1 immune or undamaged Th1 It closes, is increased it is thus determined that indicant at least partly indicates that Th1 is immune.In the representative example of these embodiments, not to Subject applies polypeptide complex or composition of the invention.

When at least one Th1 immune state biomarker includes PD-L1, for assessing the instruction of Th1 immune state Object is more reliable and has stronger diagnosis capability.Therefore, in some embodiments, Th1 immune state biomarker is come from Pair biomarker value for determining indicant.For example, in some preferred embodiments, biomarker is to being PD- L2 and PD-L1.When using the biomarker of more than one Th1 immune state in the method for the invention, the suitably party Method further includes that combination function is applied to biomarker value.In this respect, the illustrated examples of suitable combination function are selected from: Additive model, linear model, support vector machines, neural network model, Random Forest model, regression model, genetic algorithm, annealing Algorithm, weighted sum, Neighborhood Model and probabilistic model.

Preferably, method described in above-mentioned and elsewhere herein is suitable for including: that (a) determines the first Th1 immune state The biomarker value of biomarker；(b) the corresponding biomarker value of the 2nd Th1 immune state biomarker is determined； (c) indicant, the instruction are determined using the biomarker value that the first and second Th1 immune state biomarkers record Object indicates the ratio of the biomarker value of the first and second Th1 immune state biomarkers record.In such theory In bright property method, the first Th1 immune state biomarker is PD-L2, and the 2nd Th1 immune state biomarker is PD-L1.For example, in some embodiments, for the ratio of control PD-L2:PD-L1 biomarker value, sample The ratio of first and second Th1 immune state biomarker values of middle determination be (" sample Th1 immune state biomarker Ratio ") reduce, wherein the ratio of the control PD-L2:PD-L1 biomarker value with there are normal Th1 it is immune or not by The immune correlation of the Th1 of damage, and determine that the indicant at least partly indicates Th1 immunocompromised host.For example, suitably, sample The ratio of biomarker value be no more than control biomarker value ratio (for example, from normal Th1 immune response or In the resulting control sample of subject of undamaged Th1 immune response determine) about 95%, 94%, 93%, 92%, 91%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% (and each integer therebetween).

On the contrary, the ratio relative to control PD-L2:PD-L1 biomarker value, when sample P D-L2:PD-L1 biology mark When remembering that the ratio of object value improves, determine that indicant at least partly indicates that Th1 is immune and increases, wherein the control PD-L2:PD- The ratio of L1 biomarker value is immunized to there are normal Th1 or undamaged Th1 is immune related.In these cases, sample The ratio of product PD-L2:PD-L1 biomarker value is at least the ratio for compareing biomarker value (for example, normal from having In the resulting control sample of subject of Th1 immune response or undamaged Th1 immune response determine) about 105%, 106%, 107%, 108%, 109%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% (and between Each integer between the two).In other embodiments, when the ratio of sample P D-L2:PD-L1 biomarker value with it is right According to PD-L2:PD-L1 biomarker value ratio it is roughly the same when, determine indicant at least partly not indicate normally or not Impaired Th1 is immune, wherein the ratio of the control PD-L2:PD-L1 biomarker value with there are normal Th1 it is immune or The immune correlation of undamaged Th1.In these cases, the ratio of sample P D-L2:PD-L1 biomarker value is usually to compare The ratio of biomarker value is (for example, from the subject with normal Th1 immune response or undamaged Th1 immune response In resulting control sample determine) about 96% to 104% (and all integer percents therebetween).In these embodiment party In the representative example of case, polypeptide complex or composition of the invention are not applied to subject.

Any known technology for measuring protein biomarkers object or nucleic acid biomarker object is suitable for the present invention.Example Such as, flow cytometry, immunoassays, mass spectrum, microarray dataset, array and hybridization platform or combinations thereof can be used to measure biology Marker.

The discovery of the present inventor make it possible to realize diagnosis have undesirable Th1 immune state disease and/or illness ( Also be interchangeably referred to as " Th1 related disease " or " Th1 associated disease " herein) method and for applying therapeutic scheme, The therapeutic scheme includes polypeptide complex or composition of the invention for treating these diseases.

Therefore, on the other hand, the present invention provides method described in above and elsewhere herein, wherein the indicant For diagnosing the existence or non-existence of Th1 related disease or illness.In some embodiments, the disease or illness and reduction Or inhibition Th1 immune state it is related, and when PD-L2 level is lower than predetermined threshold from the sample that subject obtains, Diagnose the disease or illness.For example, disease relevant to reduction or inhibition Th1 immune state or illness can be cancer (including metastatic cancer) or pathogenic infection.

Detailed description of the invention

Fig. 1 is the graphical representation output from facs analysis, characterizes the expression of biomarker on cell surface.On DC PD-L2 expression it is negatively correlated with malarial parasite mass formed by blood stasis in the mankind.(A-C) 7 Healthy People volunteer inoculating plasmodium falciparums (P.falciparum), the CD11c that (A) PD-L1 and (B) PD-L2 are expressed in blood and before infecting was checked with 7 days after infection⁺DC Percentage.(C) figure shows the number of parasites of every milliliter of blood and the ratio of %PD-L2:%PD-L1DC.R101 is to R108 generation The each volunteer of table.P value examines the null hypothesis that global slopes are zero.

The diagram of Fig. 2 shows the parasitemia average percent of typical course of infection in (A) mouse, and mouse infection has non- 17XNL plants of malaria of lethal Xia Shi plasmodium (P.chabaudi) or Plasmodium yoelii (P.yoelii), and monitor and be up to 40 days. (B) posting for typical case's course of infection in the mouse of YM plant of lethal Plasmodium yoelii or Bai Shi mouse plasmodium (P.berghei) is infected Infested mass formed by blood stasis average percent, and monitor 10 days.Error bars indicate SEM (n=4-8).

The diagram of Fig. 3 shows total CD11c of (A) expression PD-L1⁺The percentage of DC, and (B) PD-L1⁺CD11c⁺On spleen DC The average fluorescent strength (MFI) of surface PD-L1 expression, mouse of the spleen DC from initial (naive) and infection is (after infection 7 days).(C) total CD11c of PD-L2 is expressed⁺The percentage of DC, and (D) come from initial and infecting mouse (after infection 7 days) PD- L2⁺CD11c⁺The MFI of spleen DC upper surface PD-L2 expression.Bar shaped on scatter plot indicates average value.Pass through Wilcoxon matching pair Signed rank test analyzes the conspicuousness between matched 0th day and the 7th day human sample.It is more using single factor test ANOVA and Tukey (p < 0.05 * of the conspicuousness between multiple groups is analyzed in comparing check again；**p<0.01；***p<0.001；P < 0.0001 * * * is used for Comparison among groups).(A) and the data of (C) indicate that combined independent experiment, independent experiment obtain similar result.

The diagram of Fig. 4 shows PD-L1 and PD-L2 expression on the DC from lethal and non-lethality malaria.(A)- (E) from 17XNL plants of (A) initial mouse and bacterial strain of being infected with malaria (B) non-lethality Plasmodium yoelii or (C) non-lethality summer The work CD19 of the mouse of family name plasmodium, YM plants of (D) lethal Plasmodium yoelii or (E) lethal Bai Shi mouse plasmodium^-CD3^- D11c⁺The fluidic cell spectrum of upper PD-L1, PD-L2 and CD8 expression of DC.(F) used in Fig. 3 B and D, in different voltages setting It is not measured on flow cytometer, expresses PD-L1's and PD-L2 from initial mouse and infecting mouse (after infection 7 days) CD11c⁺The MFI of spleen DC repeats to test.(G) from be isolated from DC (it is initial or the 7th day with 17XNL plant of Plasmodium yoelii with YM plants infection) RNA in PD-L1 and PD-L2 quantitative RT PCR analysis.By result to three house-keeping genes (CxxC1, TBP and MRPL13A geometrical mean) is normalized.Numerical value is expressed as the average value ± SEM of independent experiment three times, and is expressed as Relative to the result for being uninfected by mouse.

The diagram display PD-L2 of Fig. 5 improves immunity and survival to malaria infection.(A-C) for (A) PD-L2ko and In the wild-type mice that wild-type mice (n=4) or (B) use rat IgG or anti-PD-L2 blocking antibody (n=5) to treat, about The average value of parasitemia percentage in the canonical process of 17XNL plants of malaria of family name plasmodium；And (C) with rat IgG or resists In the wild-type mice of PD-L2 blocking antibody treatment (n=5), parasitemia in the canonical process of Xia Shi plasmodium malaria The average value (on a log scale) of percentage.Arrow indicates that helminth removing is controlled than anti-PD-L2 in the mouse of rat IgG treatment The mouse for the treatment of is 4 days early.Data represent one of two independent experiments for obtaining similar results.Use the nonparametric based on bilateral The conspicuousness at Mann-Whitney U check analysis certain time points.Error bars indicate (p < 0.05 * SEM；**p<0.005).

During the diagram of Fig. 6 is shown in 17XNL plants of malaria of Plasmodium yoelii, it is immune that PD-L2 adjusts protection, symptom and Th1. (A-D) rat IgG or anti-PD-L2 is used to block property in (A)-(B) wild-type mice and PD-L2ko mouse (n=5) or (C)-(D) In the wild-type mice of antibody (n=5) treatment, during the typical case of 17XNL plants of malaria of Plasmodium yoelii, from Fig. 5 A and B weight The parasitemia and symptom scores tested again.(E) it in the experiment of repetition described in Fig. 2 C, is hindered with rat IgG or anti-PD-L2 In the wild-type mice of disconnected property antibody (n=5) treatment, the parasitemia during Xia Shi plasmodium malaria canonical process.Arrow Indicate that helminth is removed 3 days more early than the mouse that anti-PD-L2 is treated in the mouse of rat IgG treatment.

The diagram of Fig. 7 shows (A) for by hybridoma supematant assesse CD4⁺T in T cell_betThe gating strategy of expression. (B)-(C) scatter plot show in the wild-type mice treated with rat IgG (n=7) or anti-PD-L2 blocking antibody (n=7), Or the number of T cell in every spleen in 14 days PD-L2ko mouse (n=3) is infected with 17XNL plants of Plasmodium yoelii.(B) every spleen Dirty middle expression T_betCD4⁺CD62L^hiOr CD4⁺CD62L^loThe average number of T cell.(C) in the presence of initial DC, to parasitism Worm antigen (MSP1₁₉) generate response and the CD4 in every spleen of secretion of gamma-IFN in ELISPOT culture⁺The average of T cell Mesh.(D) scatter plot is shown, at the 14th day, is generated response to helminth peptide (Pb1) in the presence of initial DC and is trained in ELISPOT CD8 in every spleen of secretion of gamma-IFN in feeding object⁺The number of T cell.In addition to PD-L2ko mouse assessment one secondly, data come from 2 A independent experiment.Use nonparametric Mann-Whitney U check analysis conspicuousness (p < 0.05 * based on bilateral；***p< 0.001)。

The diagram of Fig. 8, which is shown in the mouse of Plasmodium yoelii 17XNL plants of infection, blocks PD-L2 to inhibit helminth specificity CD4⁺The amplification of T cell.Property is blocked with 17XNL plants of infection wild-type mices of Plasmodium yoelii, and with rat IgG or anti-PD-L2 Antibody (n=7) treatment.(A), (B), (C) express T in every spleen on the 14th day at (A) the 0th day, (B) the 7th day and (C)_betCD4⁺CD62L^hiOr CD4⁺CD62L^loThe number of T cell；(D) to parasite antigen (MSP1 in the presence of initial DC₁₉) generate response And in ELISPOT culture secretion of gamma-IFN (IFN-γ) CD4⁺T cell number.(E) it is measured by incorporation EdU, To parasite antigen MSP1 in the presence of initial DC₁₉The CD4 for generating response and being proliferated in culture⁺T cell number；(F) and (G) The average level of (F) IFN-γ and (G) IL-10 in 17XNL plants of infected mouse seras of Plasmodium yoelii.(H) every spleen expression The CD4 of CD25 and FoxP3⁺The average number of T cell (regulatory T cells).Item on scatter plot indicates intermediate value.Data represent two A combined independent experiment.Use nonparametric Mann-Whitney U check analysis conspicuousness (p < 0.05 * based on bilateral；**p <0.01；***p<0.001).

The PD-L1 and PD-L2 of the diagram display DC expression of Fig. 9 are to T cell and immune different role.In lethal Yue Shi YM plants of plasmodium acquire, and are directed to the marker (CD4 of DC subgroup for infection the 7th day⁺；CD8⁺；B220⁺pDC；And CD11b⁺DC) into Line flag comes from the CD19 of (A) wild type and (B) PD-L1ko mouse^-CD3^-CD11c⁺The fluidic cell of DC is composed.(C) survival is bent Line is displayed without protective effect of the DC of PD-L1 expression to lethal malaria.With 10⁴The lethal agent of YM plants of pRBC of Plasmodium yoelii Amount infection wild type and PD-L1ko mouse, separates DC from (cured substance) mouse of infection, and by 1x10⁷A DC transfer Into every mouse of every group four initial mouse, experiment is duplicate.After 24 hours, every mouse is with 10⁴Plasmodium yoelii YM plants of pRBC infection, the survival of monitoring in every 1 to 3 day continue 50 days.(D)-(E) it repeats to test, wherein in lethal Plasmodium yoelii The 7th day of YM plants of infection acquires, and infusion has about 1 × 10 from wild type and PD-L1ko mouse⁷Posting in the initial mouse of DC Infested mass formed by blood stasis.After 24 hours, with 10⁴YM plants of pRBC of Plasmodium yoelii infect the mouse of every infusion, and every 1 to 3 day is supervised It surveys, continues 50 days.The result is that average value ± SEM, n=4 mouse/group.(I) survival curve shows PD-1ko mouse to lethal Malaria has immune.In duplicate experiment, the group lethal 10 of five wild types and PD-1ko mouse⁴Plasmodium yoelii YM plants of pRBC are infected, and the survival of monitoring in every 1 to 3 day continues 50 days.(J)-(K) it repeats to test, wherein with 10⁴Yue Shi malaria is former Parasitemia in the wild type and PD-1ko mouse of YM plants of pRBC of worm infection monitors, continues 50 days for every 1 to 3 day.The result is that Average value ± SEM, n=5 mouse/group.(L)-(P) with the CD4 of the DC culture of expression PD-L1 and PD-L2⁺CD62L^lo PD-1⁺ The flow cytometry of CD3 and ICOS expression in T cell；Wherein (L) is the negative control that DC is only free of comprising T cell；(M) It is the positive control comprising T cell and DC；(N) blocking of PD-1；(O) blocking of PD-L1；The blocking of (P) PD-L2；36 is small Shi Hou.It is infected from 17XNL plants of Plasmodium yoelii in 12-14 days mouse spleens and separates T cell and DC.Based in anti-PD-L1 training Support the door that the clear bimodal selection found in object determines high CD3 or ICOS expression.(Q) scatter plot is shown in independent real from three The CD4 that (n=3 to 5) there is high CD3 and ICOS to express in the repeating hole tested⁺CD62^loPercentage/hole of T cell, three independences Experiment is shown as white, light blue and navy blue point.Error bars indicate average value.Match using from the non-of one of three experiments (unilateral side) is examined to analyze conspicuousness (p < 0.05 * t-；**p<0.005；***p<0.0005；****p<0.0001).

The diagram of Figure 10 is shown in the patient with metastasis melanin tumor PD-L2 expression on blood DC and reduces.(A-C) Blood suffers from the patient of metastasis melanin tumor from 8 Healthy People volunteers, 4 with non-black melanoma lesion and 4.Inspection It looks into their blood and expresses the CD11c of (A) PD-L1 and (B) PD-L2⁺The percentage of DC.(C) figure shows %PD- in every group The ratio of L2:%PD-L1DC.Using the P value of Mann Whitney (A) and (B) examined between each group, (C) passes through Kruskal-Wallis multiple comparative test is calculated.

Eight dimer forms of the diagram display PD-L2 of Figure 11 prevent lethal malaria.(A) mean percent of parasitemia Than；(B) logarithmic scale shows the number of the mouse with parasitemia；(C) survival (x-axis indicates metainfective number of days)；(D) 3,5 and 7 days after infection, the Plasmodium yoelii YM in the wild-type mice treated with negative control (people) IgG or dimer PD-L2 The parasitemia percentage of strain malaria canonical process.And (E) after parasitemia is detectable, on day 3 followed by the In 5 days and the 7th day (n=12 in total, from the independent experiment three times) wild-type mices with control (people) IgG or PD-L2 treatment The symptom scores of YM plants of malaria canonical process of Plasmodium yoelii (x-axis shows metainfective number of days).Allow the small of all survivals Mouse rest, and after 150 days with YM plants of malaria of the lethal Plasmodium yoelii of same dose (not applying other PD-L2) again Attack, together with the new control mice (control Ig-R) of age-matched.(F) 20 days after being attacked again with YM plants of Plasmodium yoelii, Give peak percentage (the x-axis display sense of parasitemia in the initial mouse of the 200 μ L blood from PD-L2 treatment mouse Number of days after dye).The low number of helminth in the testing inspection donor mice blood.(G) symptom scores, (H) survival (x Axis indicates metainfective number of days), and (I) the 3rd, 5 and 7 day after infection (n=9 in total comes from 2 independent experiments), with control In the wild-type mice of (people) IgG or PD-L2 treatment in Bai Shi mouse Infected With Plasmodium canonical process parasitemia average hundred Divide than (x-axis shows metainfective number of days).Error bars indicate SEM.It is survived using Log-rank (Mantel-Cox) check analysis Conspicuousness.

Figure 12 shows ten dimer PD-L2 (sPD-L2) of solubility to the diagram of the protective effect of advanced melanoma.By 6 The group of C57BL/6 mouse is subcutaneously implanted 5 × 10⁵A B16.F0 melanoma cells.At the 9th day or so, when average tumor ruler Very little is about 100mm³When, 200 μ g human IgG of mouse or sPD-L2, every 1-2 days monitoring tumor sizes were given at the 9th, 11 and 13 day. Data indicate one in two independent experiments merged, and two independent experiments obtain similar result.Using based on unilateral side Nonparametric Mann-Whitney U examine and determine P value (* * * p=0.0006 is for the comparison between group).

Figure 13 shows sPD-L2 to the diagram of the early protection of melanoma.The group of 6 C57BL/6 mouse is subcutaneously implanted 1×10⁵A B16.F10 melanoma cells.At the 9th day or so, when Mean tumor sizes are about 100mm³When, in the 3rd, 9 and 15 It gives 200 μ g human IgG of mouse or sPD-L2, every 1-2 days monitoring tumor sizes.Data indicate in two independent experiments merged One, two independent experiments obtain similar result.It is true using being examined based on unilateral nonparametric Mann-Whitney U Determine P value (* * * p=0.03 is for the comparison between group).

Specific embodiment

1. definition

Unless otherwise defined, otherwise all technical and scientific terms used herein have with it is of the art general The logical identical meaning of the normally understood meaning of technical staff.Although with those of be described herein similar or equivalent any method and Material practice for use in the present invention or test, but describe preferred method and material.For purposes of the present invention, following art Language is defined as follows.

Article " a kind of (a) " used herein and " a kind of (an) " refer to that one or more than one (i.e. at least one) is preced with Word grammar object.For example, " a kind of element " indicates an element or more than one element.

As it is used herein, "and/or" refers to and covers any and all of one or more related listed items Possible combination, and substitution (or) explain when, lack combination.

In addition, term " about " as used herein and " approximation ", when be related to measurable magnitude (such as amount, dosage, the time, temperature, Activity, level, number, frequency, percentage, size, size, amount, weight, position, length etc.) when, it is meant that including specified amount, Dosage, time, temperature, activity, level, number, frequency, percentage, size, size, amount, weight, position, length etc. ± 15%, ± 10%, ± 5%, ± 1%, ± 0.5% or even ± 0.1% variation.In term " about " and " approximation " and with reference to more In the case that positioning or position in peptide are used in combination, these terms include ± up to 20 amino acid residues, ± up to 15 Amino acid residue, ± up to 10 amino acid residues, ± up to 5 amino acid residues, ± up to 4 amino acid residues, ± it is more Up to the variation of 3 amino acid residues, ± up to 2 amino acid residues or even ± 1 amino acid residue.

Term " (concurrently) is administered simultaneously " or " being administered simultaneously " or " co-administration " etc. refer to that application contains two The single composition of kind or more active material, or applied every kind of active material as separated composition, and/or In time short enough synchronous (contemporaneously) or simultaneously (simutaneously) or pass sequentially through separated way Diameter is delivered, so that effectively result is equal to the result obtained when all these active materials are applied as single composition.It is " same When " refer to that activating agent is substantially simultaneously applied, and ideally applied together in same preparation." synchronization " refers to that activating agent exists Close application on time, such as a kind of medicament are applied in about one minute to about one day before or after another medicament.It is any Time simultaneously is all useful.However, it is often the case that medicament will be in about 1 minute to about 8 when not being administered simultaneously Application in hour, and suitably applied in less than about 1 to about 4 hour.When synchronous application, the medicament is suitably tested The same area of person is applied.Term " same area " includes exact position, but can be in about 0.5 to about 15 centimetre, preferably In about 0.5 to about 5 centimetre.The term as used herein " separated " refer to medicament interval apply, such as with about one day to it is a few week or It applies at the interval of some months.Activating agent can be applied in any order.The term as used herein " sequentially " refers to medicament successively Application, such as with several minutes, a few hours, one or more interval applications of a couple of days or several weeks.It if appropriate, can be in rule Administering active agents in repetitive cycling.

Terms used herein " adjuvant " refer to work as to be combined with specific immunogens (for example, polypeptide complex of the invention) In use, the compound of resulting immune response (for example, Th1 immune response) will be enhanced, including strengthen or expand antibody and thin The specificity of one or both of born of the same parents' immune response.

Term " medicament " or " regulator " include the compound for inducing required pharmacology and/or physiological role.The term It further include the pharmaceutically acceptable and pharmacologically active principles of those of specifically mentioned compound herein, including but not limited to Salt, ester, amide, prodrug, active metabolite, analog etc..When using above-mentioned term, it should be understood that it include activating agent itself with And pharmaceutically acceptable pharmacological activity salt, ester, amide, prodrug, metabolin, analog etc..It cannot narrowly explain art Language " medicament ", but extend to small molecule, protein molecular (such as peptide, polypeptide and albumen) and comprising their compositions and Genetic molecule (such as RNA, DNA and analogies) and its chemical analog and cell reagent.Term " medicament " includes that can generate With the polynucleotides of the cell for secreting polypeptide described herein, and the nucleotide sequence comprising encoding polypeptide.Therefore, term " medicine Agent " extends to nucleic acid construct, including carrier, such as virus or non-virus carrier, expression vector and in cell context The plasmid of expression and secretion.

As used herein, refer to can be with spy for term " antigen " and its grammatically equivalent expression (for example, " antigenicity ") Compound, composition or the substance that anisotropic body fluid or cellular immunity product specificities combine, for example, antibody molecule or T cell by Body.Antigen can be any kind of molecule, including such as haptens, simple intermediate metabolites, sugar (such as oligosaccharides), lipid With hormone and macromolecular such as complex carbohydrates (such as polysaccharide), phosphatide and albumen.The antigen of common class include but It is not limited to viral antigen, bacterial antigens, fungal antigen, protozoan and other parasite antigens, tumour antigen, itself is participated in and exempts from Antigen, toxin and other various antigens of epidemic disease, allergy and graft rejection.

" antigen binding molecules " refer to the molecule for having binding affinity to target antigen.It should be understood that the term extends to Show the immunoglobulin, immunoglobulin fragment and non-immunoglobulin derived protein frame of antigen-binding activity.It can use In implement representative antigen binding molecules of the invention include polyclonal and monoclonal antibody and their segment (such as Fab, Fab'、F(ab')₂, Fv), single-stranded (scFv) and domain antibodies (including such as shark and camellid antibody) and comprising anti- The fusion protein of body, and any other modification configuration comprising antigen binding/recognition site immunoglobulin molecules.Antibody Antibody including any classification, such as IgG, IgA or IgM (or its subclass), and antibody needs not be any particular category.It takes Certainly in the antibody amino acids sequence of its heavy chain constant region, immunoglobulin can be assigned to different classifications.Immunoglobulin has Five major class: IgA, IgD, IgE, IgG and IgM, some of them can be further divided into subclass (isotype), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.Heavy chain constant region corresponding to different immunoglobulin class is referred to as α, δ, ε, γ and μ. The subunit structure and 3-d modelling of different classes of immunoglobulin are well-known.Antigen binding molecules further include dimer The antibody of antibody and multivalent forms.In some embodiments, antigen binding molecules are chimeric antibodies, wherein heavy chain and/or A part of light chain with derived from particular species or belong to corresponding sequence in the antibody of specific antibodies classification or subclass it is identical or It is homologous, and the chain rest part to derived from another species or belong to corresponding sequence in the antibody of another antibody isotype or subclass The segment that identical or homologous and antigen binding molecules further include such antibody is arranged, is lived as long as they show required biology Property is (see, e.g. U.S. Patent number 4,816,567；And Morrison et al., 1984, Proc.Natl.Acad.Sci.USA 81:6851-6855).Humanized antibody is also contemplated, usually by that will exempt from from inhuman (for example, rodent, preferably mouse) The heavy chain of epidemic disease globulin and the complementary determining region (CDR) of light chain variable chain are transferred in people's variable domains and generate.Then exist Replace the Typical residues of human antibody in the framework region of inhuman counterpart.Using be originated from humanized antibody antibody component eliminate with The relevant potential problems of the immunogenicity of non-human constant region.For cloning inhuman (especially mouse) immunoglobulin variable domain domain General technology be described in such as Orlandi et al. (1989, Proc.Natl.Acad.Sci.USA 86:3833).For generating The technical description of Humanized monoclonal antibodies is in such as Jones et al. (1986, Nature 321:522), Carter et al. (1992, Proc.Natl.Acad.Sci.USA 89:4285), Sandhu (1992, Crit.Rev.Biotech.12:437), Singer et al. (1993, J.Immun.150:2844), Sudhir (compile, Antibody Engineering Protocols, Humana Press,Inc.1995)、Kelley(“Engineering Therapeutic Antibodies,”in Protein Engineering:Principles and Practice Cleland et al. is compiled, 399-434 pages (John Wiley&Sons, Inc.1996) and Queen et al., U.S. Patent number 5,693,762 (1997).Humanized antibody includes " primatized " anti- Body, wherein the antigen binding domain of antibody is derived from the antibody generated and with interest antigen immunizing macaque monkeys.It also considers as anti- Former binding molecule is humanized antibody.

Term " antigen presenting cell " (APC) is to refer in order to avoid epidemic disease systemic characteristic effector cell (herein also referred to as " exempts from Epidemic disease effector cell " or " IEC ") peptide-MHC composite form of identification presents one or more antigens, thus adjust (for example, Stimulation/enhancing or reduction/tolerance/are not replied) to the cell class of the immune response of presented antigen.In specific reality of the invention It applies in scheme, APC can activate IEC, such as T lymphocyte, including CD8⁺And/or CD4⁺Lymphocyte.There is potential work in vivo It not only include such as sole duty APC (such as dendritic cells, macrophage, Langerhans cell, monocyte for the cell of APC effect And B cell), it further include non-full-time APC, illustrated examples include that the epithelial cell of activation, fibroblast, neuroglia are thin Born of the same parents, pancreatic beta cell and vascular endothelial cell.The cell of many types can present antigen for IEC (packet in its cell surface Include T cell) identification.

Term " biomarker " typically refers to measurable feature, reflects depositing for physiology and/or pathological and physiological condition Or property (for example, severity or state), the indicant of the risk including developing into specific physiology or pathological and physiological condition. It is obtained for example, biomarker can reside in from the subject before physiology or pathological and physiological condition (including its symptom) breaking-out Sample in.Therefore, the presence of biomarker may indicate that risk increases from the sample that subject obtains, i.e. subject The risk of physiology or pathological and physiological condition or its symptom will be developed into.Alternatively, in addition, biomarker can be in individual Normal expression, but its expression may change before physiology or pathological and physiological condition (including its symptom) breaking-out (that is, it is improved (up-regulation；Overexpression) or reduction (downward；Low expression)).Therefore, the horizontal variation of biomarker may indicate that increased wind Danger, i.e., subject would develop into physiology or pathological and physiological condition or the risk of its symptom.Alternatively, in addition, biomarker Horizontal variation can reflect the variation of specific physiology or pathological and physiological condition or its symptom in subject, thus allow physiology or The property (for example, severity) of pathological and physiological condition or its symptom are tracked a period of time.This method can be used for for example monitoring Therapeutic scheme is to assess the purpose of its validity (or other aspect) in subject.As described herein, biomarker is referred to The level of object includes the activity of the concentration of biomarker or the expression of biomarker or biomarker, below will More detailed description.

Term " biomarker value " refers to biomarker measurement corresponding for at least one of subject or obtains Value, and its abundance or concentration for indicating to be derived from biomarker in the sample of subject typically at least in part.Therefore, raw Substance markers object value can be the biomarker of measurement value (it is the value of biomarker measured for subject) or Person can be the value of the biomarker of derivation (derived), and (it is pushed away from the value of biomarkers of one or more measurement The value led, such as the value of the biomarker by the way that function to be applied to one or more measurements).Biomarker value can By be it is any it is appropriate in the form of, be specifically dependent upon the mode of determining numerical value.It is, for example, possible to use high-throughput techniques to determine biology mark Remember object value, for example, mass spectrum, microarray dataset, array and hybridization platform, immunoassays, flow cytometry or these technologies it is any Combination.In a preferable example, biomarker value is related to producing using the quantitative protein expression of the technologies such as such as flow cytometry The activity level or abundance of object or other measurable molecules.In this case, biomarker value can be table in the sample It, as the skilled person will appreciate and will in further detail below up to the form of the percent value of the cell of biomarker Ground description.

Term " biomarker spectrum " refers to that one or more biomarkers are (for example, polypeptide in one or more types Molecule, cDNA molecule etc.) or its refer to (indication), and, for example, can measure aspect biomarker characteristics (for example, Biomarker value).Biomarker spectrum may include single creature marker integral level, abundance or amount, with subject's Th1 immune state is related (for example, Th1 immune state or reduced Th1 immune state of enhancing).Alternatively, biomarker is composed It may include biomarker as at least two or its reference, wherein biomarker can be identical or different class Not, such as polypeptide and nucleic acid.Therefore, biomarker spectrum may include at least 1,2,3,4,5,6,7,8,9,10,11,12,13, 14、15、16、17、18、19、20、21、22、23、24、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95 100 or more biomarker or its reference.In some embodiments, biomarker spectrum include it is several, several, tens Kind or hundreds of biomarkers or its reference.Biomarker spectrum can also include one or more controls or internal standard. In certain embodiments, biomarker spectrum includes at least one biomarker as internal standard or its reference.? In other embodiments, biomarker spectrum includes the reference of the biomarker of one or more types.It is used herein Term " reference " only refer to such case, i.e. biomarker spectrum symbol, data, abbreviation or other classes for including biomarker Like mark, rather than the entity of biomarker molecule itself.Terms used herein " biomarker spectrum " is also used for referring to biology The combination of marker value or at least two biomarker values, wherein individual biomarker value corresponds to from one or more Value that is that subject can measure or deriving gained biomarker, a combination thereof are characterized in that Th1 immune state, discrete shape State, illness classification, illness hypotype or prognosis discrete state, illness classification, illness hypotype.Term " spectrum biomarker " is for referring to The subset of biomarker has been accredited and has composed for biomarker, can be used for carrying out clinical assessment, for example, for being included in or Exclude the different classifications or severity, the hypotype of different syndromes or different prognosis of particular condition, illness.Compose biomarker Number will be different, but usually 10 or smaller magnitude.

When referring to molecule, term " chimeric " refer to the molecule contain be derived from, obtain or be isolated from or based on two kinds or The part in more kinds of Different Origins or source.Therefore, when polypeptide includes two or more amino acid sequences of separate sources, And the polypeptide sequence that cannot be found together in nature including (1) is (that is, at least one amino acid sequence and other amino acid At least one of sequence is heterologous) or the adjacent amino acid sequence of (2) non-natural, then the molecule is chimeric.

" aggregation (clustering) " used herein and grammatical equivalents refer to more than two identical Th1 immune states Any reversible or irreversible association of biomarker (for example, PD-L2).Cluster (cluster) can by 3,4,5,6,7, 8, the biomarkers composition such as 9,10,12,20.The cluster of two biomarkers is referred to as dimer.Three or more lifes The cluster of substance markers object is commonly known as oligomer, and wherein the number of individuals of cluster has its respective reference, such as three biologies The cluster of marker is tripolymer, and the cluster of four biomarkers is the tetramer, and the cluster of five biomarkers is five poly- Body, the cluster of six biomarkers are six aggressiveness, and the cluster of seven biomarkers is heptamer, eight biomarkers Cluster is eight aggressiveness, and the cluster of nine biomarkers is nine aggressiveness, and the cluster of ten biomarkers is ten aggressiveness, 12 The cluster of biomarker is that the cluster of ten dimers and 20 biomarkers is 20 aggressiveness.

" coded sequence " refers to the final mRNA product of the polypeptide product or gene that facilitate encoding gene (for example, montage The mRNA product of gene afterwards) any nucleic acid sequence.On the contrary, term " non-coding sequence " refers to the polypeptide product to encoding gene Or the final mRNA product of gene does not have contributive any nucleic acid sequence.

Term " coiled coil " or " coiled-coiled structure " herein are used interchangeably, the structural motif in finger protein, Two of them or more alpha-helix (most commonly 2-7 alpha-helix) is crimped into place, as rope chain (dimer and Tripolymer is the most common type).Many coiled coil type albumen participate in important biological function, such as the tune of gene expression Save such as transcription factor.Coiled coil generally but not always includes the repeat pattern of hydrophobicity (h) and polarity (p) amino acid residue Hpphppp or hppphpp, referred to as heptapeptide repeat (referring to hereinbelow).Sequence with this repeat pattern is folded into α- Helix secondary structure causes hydrophobic residue to be rendered as " striped ", and striped is gently wound around spiral with left-handed spiral Amphiphilic structure.In water-filled environment, the most advantageous manner of two such helical arrangements is to wrap hydrophobic chain opposite to each other It wraps up in and is clipped between hydrophilic amino acid.Therefore, burying for hydrophobic surface provides thermodynamics driving for the oligomerization of alpha-helix Power.Packaging in coiled-coil interface is closely.Alpha-helix can be parallel or antiparallel, and generally use left-handed Supercoil.Although be not it is highly advantageous, nature and design albumen in also observe a small amount of dextrorotation coiled coil.Based on known Common sense, term " coiled coil " or " coiled-coiled structure " are clear to those skilled in the art.It is outstanding in this respect It refers to the paper in relation to coiled-coiled structure, such as Cohen and Parry (1990.Proteins 7:1-15)；Kohn and Hodges (1998.Trends Biotechnol16:379-389)；Schneider et al. (1998.Fold Des 3:R29- R40)；Harbury et al. (1998.Science 282:1462-1467)；Mason and Arndt (2004.Chem-BioChem 5:170-176)；Lupas and Gruber (2005.Adv Protein Chem 70:37-78)；Woolfson(2005.Adv Protein Chem70:79-112)；Parry et al. 2008.J Struct Biol 163:258-269)；And Mcfarlane Et al. (2009.Eur J Pharmacol 625:101-107).

As used herein, refer to " with diagnosis " for identifying the subject susceptible to particular treatment or for monitoring treatment And/or identify the diagnostic method and/or reagent of subject or subject's subgroup or other group's effective doses.For the mesh of this paper , refer to reagent with diagnosis, such as determining Th1 immune system biomarker in sample (for example, as described herein) Biomarker value reagent.The test for referring to reagent with diagnosis and being carried out with reagent.

As used herein, term " complementation " and its grammer equivalent expressions refer to two or more structural details (for example, Peptide, polypeptide, nucleic acid, small molecule or part thereof etc.) can hybridize, oligomerization is (for example, dimerization, trimerizing, tetramerization, five poly- Change, six dimerizations, seven dimerizations, eight dimerizations, nine dimerizations, ten dimerizations, 11 dimerizations, ten dimerizations), interaction or otherwise The feature of compound is formed each other.For example, " complementary region of polypeptide " can get together to form compound.

As used herein, term " compound " refers to directly with one another and/or the molecule of mediate contact is (for example, peptide, polypeptide Deng) assembly or aggregation.In specific embodiments, " contact " or more specifically " directly contact " mean two or more Multiple molecules are close enough so that attractive noncovalent interaction, such as Van der Waals force, hydrogen bond, ion and hydrophobic phase The interaction of molecule is dominated in interaction etc..In such an implementation, the compound of molecule is formed in such a situa-tion (for example, peptide and polypeptide), so that compound is thermodynamically advantageous (for example, with the non-agglomerated of its ingredient or non-multiple Conjunction state is compared).Terms used herein " polypeptide complex " or " albumen composition " refer to tripolymer, the tetramer, pentamer, Six aggressiveness, heptamer, eight aggressiveness, nine aggressiveness, ten aggressiveness, 11 aggressiveness, ten dimers or more advanced oligomer.Specific In embodiment, it is self-assembly of polypeptide complex by chimeric polyeptides, the chimeric polyeptides include PD-L2 polypeptide and at least one A oligomerization domain.

Throughout the specification, it indicates unless the context otherwise, otherwise word "include", "comprise" and " containing " will be managed Solution is not excluded for any other step or element or step to imply the group including the step or element or step or element Or the group of element.Therefore, the element that the use of term " includes " etc. indicates listed is required or enforceable, but other Element is optional and may exist or be not present." consist of " mean include and be limited to phrase " consist of " it Content afterwards.Therefore, the element that phrase " consist of " indicates listed is required or compulsory, and other are not present Element." substantially by ... form " refers to any element including listing after phrase, and is limited to not interfere or facilitate The other elements of the activity or effect specified in the disclosure for listed element.Therefore, phrase " substantially by ... form " indicates Listed element is required or enforceable, but other elements are optional, and may exist or be not present, this takes Certainly the activity or effect of listed element whether are influenced in them.

As used herein, term " conjugation ", " connection ", " fusion " or " fusion " and its grammatical equivalents, two Kind or more element or component or structural domain (including chemically conjugated or recombination form is (for example, pass through heredity in any manner Fusion)) link together in the case where, be used interchangeably.Chemically conjugated method (for example, using heterobifunctional crosslinker) is It is known in the art.More specifically, as used herein, PD-L2 polypeptide-oligomerization domain fusion or conjugate refer to PD-L2 Polypeptide is hereditary or chemically conjugated at least one oligomerization domain.In specific embodiments, at least one oligomerization Structural domain is fused to PD-L2 polypeptide, such as glycine-serine (gly-ser) connector by peptide linker indirectly.In other implementations In scheme, at least one oligomerization domain directly with PD-L2 peptide fusion.

" conserved amino acid substitution " is the amino that wherein amino acid residue is replaced by the amino acid residue with similar side chain Acid replaces.The amino acid residue families with similar side chain have been defined in this field, usually can be sub- according to following progress Classification:

Table 1

The subclassification of amino acid

Conserved amino acid substitution further includes the grouping based on side chain.For example, the amino acid group with aliphatic lateral chain is sweet Propylhomoserin, alanine, valine, leucine and isoleucine；Amino acid group with aliphatic-hydroxy side chains is serine and Soviet Union Propylhomoserin；Amino acid group with beta-branched side is asparagine and glutamine；Amino acid group with beta-branched side is Phenylalanine, tyrosine and tryptophan；Amino acid group with basic side chain is lysine, arginine and histidine；With containing The amino acid group of sulphur side chain is cysteine and methionine.For example, can reasonably expect that with isoleucine or valine substitution Leucine similarly replaces certain with serine for threonin, or with structure related amino acid with glutamate for aspartate Amino acid will not generate big influence to the property of gained variant polypeptide.It can readily determine that amino acid by measuring its activity Change and whether generates functional polypeptide.Conservative substitution is as shown in table 2, entitled exemplary and preferred substitution.In general, it falls Entering the amino acid substitution in the scope of the invention is by selecting its substitution to maintaining the effect of following aspect to be not significantly different Come what is realized: (a) replacing the peptide backbone structure in region, (b) in the charge or hydrophobicity of target site punishment, or (c) side chain Part.After introducing substitution, the bioactivity of variant is screened.

Table 2

Exemplary and preferred amino acid substitution

Term " construct " refers to genetic recombination molecule comprising the core of one or more of separation from separate sources Acid sequence.Therefore, construct is chimeric molecule, and wherein two or more nucleic acid sequences of separate sources are assembled into single nucleic acid Molecule, and including containing any construct below: (1) nucleic acid sequence, it includes the adjustings not found in natural together Sequence and coded sequence (that is, at least one nucleotide sequence is heterologous relative at least one other nucleotide sequence) or (2) sequence of coding non-natural adjacent functional RNA molecule or protein part, or the starting subdivision that (3) non-natural is adjacent. Representative construct includes derived from any source and being capable of genome conformity or any recombinant nucleic acid molecules for independently replicating (such as plasmid, clay, virus, the polynucleotide molecule independently replicated, bacteriophage or linear or cyclic single strand or double-stranded DNA or RNA nucleic acid molecules), it includes the nucleic acid molecules for the one or more of nucleic acid molecules being operatively connected.Building of the invention Body generally includes the required element for instructing interest nucleic acid sequence to express, and the interest nucleic acid sequence is also contained in construct, example Such as target nucleic acid sequence adjusts nucleic acid sequence.These elements may include control element, such as can grasp with interest nucleic acid sequence Make the promoter of ground connection (so as to guide transcription), and also typically includes polyadenylation sequence.Of the invention certain In embodiment, construct be may be embodied in carrier.Other than the component of construct, carrier may include, such as one Or multiple selectable markers, one or more replication orgins (such as protokaryon and eukaryon starting point), at least one polyclonal position The element of point, and/or promotion construct stable integration into host cell gene group.Two or more constructs may include In single nucleic acid molecules, such as single carrier, or may be embodied in two or more individual nucleic acid molecules, such as Two or more individual carriers." expression construct " typically at least includes the control being operatively connected with interest nucleotide sequence Sequence processed.For example, in this way, the starting being operatively connected with nucleotide sequence to be expressed is provided in expression construct Son, for the expression in organism or part thereof (including host cell).For practice of the invention, structure is used to prepare and used The conventional composition and method for building body and host cell are well known to those skilled in the art, see, for example, Molecular Cloning:A Laboratory Manual, the third edition roll up 1,2 and 3, JF Sambrook, DW Russell and N.Irwin, Cold Spring Harbor Laboratory Press, 2000.

Term " correlation ", which refers to, determines a type of data with another type of data or with state (for example, Th1 exempts from Epidemic disease state) between relationship.

" corresponding to " or " corresponding to " refers to the ammonia that substantial sequence similarity or identity are shown with reference amino acid sequence Base acid sequence.In general, amino acid sequence and reference amino acid sequence at least partly will display at least about 70,71,72,73,74, 75,76,77,78,79,80,81,82,83,84,85,86,97,88,89,90,91,92,93,94,95,96,97,98,99% Or even as high as 100% sequence similarity or identity.

Term " oligomerization degree " refers to the number (n) of protein molecular unit in the polypeptide complex according to formula (I).

As it is used herein, term " diagnosis ", " in diagnosis " etc. use interchangeably herein, with include determine by Examination person by develop illness a possibility that or subject in illness presence or property.These terms further include determining disease or disease The severity of onste, and under the background of rational therapy, guiding treatment is diagnosed, initial selected, treatment including treatment Change (for example, adjustment of dosage or dosage) etc.." possibility " refers to that measurement has on the basis of given mathematical model Whether particular measurement or derivation biomarker value subject actually suffers from (or not suffering from) illness.For example, increased Possibility can be opposite or absolute, and can qualitatively or quantitatively express.For example, based on previous population research, It can be simply by the biomarker of measurement or the derivation of at least two Th1 immune state biomarkers in determining subject Object value, and subject is classified as in " a possibility that increase " classification, a possibility that determine increase.Term " possibility " is herein In can also be used interchangeably with term " probability ".Term " risk " be related to particular event in following a possibility that sometime occurring or Probability." risk stratification " refers to the known clinical risk factors of arrangement, to allow doctor by patient classification for development specified disease Basic, normal, high or highest risk.

As used herein, term " structural domain " refers to one of the molecule with common physicochemical characteristic or structure Point, such as, but not limited to hydrophobic, polarity, spherical and helix domain or for example ligand-combination, film fusion, signal transduction, carefully The properties such as born of the same parents penetrate, oligomerization.In general, structural domain have fold protein structure, have independently of albumen rest part and Retain the ability of its tertiary structure.In general, structural domain is responsible for the discrete functionality characteristic of albumen, and can add in many cases Add, remove or be transferred to the function in other albumen without losing the rest part of albumen and/or structural domain.Structural domain can be with Region or part thereof is coextensive；Structural domain can also include different, discrete molecular domains.The example of protein structure domain Including but not limited to cell or extracellular localization domain is (for example, signal peptide；SP), immunoglobulin (Ig) structural domain, extracellular Domain, cross-film (TM) structural domain and cytoplasm (C) structural domain.

As used herein, term " coding ", " coding " etc. refer to that nucleic acid provides the ability of another nucleic acid or polypeptide.Example Such as, if nucleic acid sequence can be transcribed and/or be translated to generate polypeptide, or if it can be processed to it is transcribed and/ Or translation then claims its " coding " polypeptide in the form of generating polypeptide.Such nucleic acid sequence may include coded sequence or coded sequence And non-coding sequence.Therefore, term " coding ", " coding " etc. include the transcription by DNA molecular and the RNA product that generates；By The albumen that the translation of RNA molecule generates；It is transcribed and is formed produced by RNA product and subsequent RNA product translation as DNA molecular Albumen；Or transcribed by DNA molecular with provide RNA product, RNA product it is processed with provide processing RNA product (for example, MRNA) and the RNA product of subsequent processing is translated and the albumen of generation.

" extracellular domain " is the structural domain of epicyte protein, extends to extracellular space (i.e. extracellular space).In general, Extracellular domain is the protein part that starting is contacted with surface, so as to cause signal transduction.Therefore, the born of the same parents of PD-L2 as defined herein Foreign lands refer to that PD-L2 extends to the part (extracellular domain) of extracellular space, but also include being responsible for combining corresponding receptor (such as PD-1 shorter part or its segment).Therefore, term " extracellular domain of PD-L2 or its segment " refers to form extracellular domain PD-L2 extracellular domain or its still be able to the part (that is, receptor binding domain) in conjunction with receptor.

Cause to the immune response of enveloped virus fusion protein or fusion protein compound or treat or prevent disease or Under the background of illness, the individual that " effective quantity " is directed to demand applies a certain amount of medicament, regardless of being single dose or work It is all effective for causing, treating or preventing for a part of series.Effective quantity by according to it is to be treated individual health and Physical condition, the sorting group of individual to be treated, composition formula, medical condition assessment and other correlative factors and become Change.It is expected that the amount will be fallen into relatively wide range, this can be determined by routine test.

Term " endogenous generation " refers to that the expression of biological amplifying nucleic acid and the related of biological amplifying nucleic acid expression product generate And/or secretion.In specific embodiments, biology be many cells (for example, vertebrate, preferably mammal, more preferably Primate, such as people), and nucleic acid is expressed in the cell or tissue of multicellular organism.

Term " expression " about gene order refer to genetic transcription generate RNA transcript (for example, mRNA, antisense RNA, SiRNA, shRNA, miRNA etc.), and where appropriate, by gained mRNA translation of transcript at albumen.Therefore, from the context may be used Can be clearly seen, the expression of coded sequence is produced from the transcription and translation of coded sequence.On the contrary, the expression of non-coding sequence produces It is born from the transcription of non-coding sequence.

As used herein, " fusion " albumen refers to two or more polypeptides being coupled together, and is not naturally to be stored in In coupling arrangement.

As used herein, term " gene ", which refers to, can be used in generating mRNA, antisense RNA, siRNA, shRNA, miRNA etc. Nucleic acid molecules refer to polypeptide and in some embodiments.Gene energy cannot be used for generating functional protein.Base Because may include code area and noncoding region (for example, introne, regulating element, including promoter, enhancer, termination sequence and 5' With 3' non-translational region).Gene can be " separation ", and it is natural with nucleic acid molecules to refer to that nucleic acid molecules are substantially or essentially free of The component that state usually combines.These components include other cell materials, the culture medium from recombinant production, and/or are used for The various chemicals of chemical synthesis nucleic acid molecules.It within its scope further include the base with continuous sequence to referring to for " gene " Cause, to limit continuous nucleic acid entity as herein defined；Or the gene of non-continuous series, thus as herein defined Limit discrete nucleic acid entity.In certain embodiments, term " gene " includes open reading frame within its scope, is compiled The non-coding nucleotide sequences of the specific polypeptide of code, introne and the neighbouring 5' and 3' that participate in Expression modulation.In this respect, the base Because can also include control sequence, such as the promoter, enhancer, termination and/or the polyadenosine that are naturally combined with given gene Polyadenylation signal or heterologous control sequences.Gene order can be cDNA or genomic DNA or its segment.It can be by channel genes For host to be maintained or introduced outside chromosome in suitable carrier.

Terms used herein " heterologous " refer to any protein part, and the selection of sequence is so that the sequence and PD-L2 are more The fusion product of peptide has the sequence different from the more propeptides of wild type PD-L2 or mature form.

Term " host " refers to any biology or its cell, either eucaryote or prokaryotes, wherein can draw Enter construct of the invention.In specific embodiments, term " host " refers to eucaryote, including single celled eukaryotic life Object, such as yeast and fungi and multi-celled eukaryotes, such as animal, non-limiting example include invertebrate (example Such as, insect, cnidaria, echinoderm, nematode etc.)；Eukaryotic parasite is (for example, malarial parasite, such as plasmodium falciparum (Plasmodium falciparum), worm etc.)；Vertebrate (such as fish, amphibian, reptile, bird, lactation are dynamic Object)；With mammal (for example, rodent, primate such as people and non-human primate).Therefore, " host is thin for term Born of the same parents " uitably include such Eukaryotic cell and derived from these Eukaryotic cell lines.Term " host cell " Within its scope further include individual cells or cell culture, can be or be any recombinant vector of the invention or point From polynucleotides recipient.Host cell includes the offspring of single host cell, and due to natural, accidental or people For mutation and/or change, offspring may be not necessarily complete with original parent cell (on morphology or in terms of total DNA is complementary) It is exactly the same.Host cell includes with recombinant vector or polynucleotides of the invention transfected or infected cell in vivo or in vitro. Host cell comprising recombinant vector of the present invention is recombinant host cell.

As used herein, term " immune effector cell " (IEC) refers to leucocyte group (including lymphocyte), in its table Effect partial receptor, such as cytokine receptor and/or Fc receptor are presented on face, passes through the receptor combination effect part (such as cell factor and/or antibody Fc district), facilitates the destruction of target cell (such as tumour cell).IEC can for example be situated between Guided cell toxicity or phagocytosis.IEC cell includes but is not limited to effector T cell, such as CD8⁺Cytotoxic T cell, CD4⁺It is auxiliary Help killing (LAK) cell of T cell, gamma delta T cells, NK cell, NK sample T cell and lymphokineactivation.The activity of IEC can be with It is adjusted by their interactions with APC, the antigen presenting cell including profession, such as macrophage, dendritic cells, Lang Ge The Chinese this cell, B cell and monocyte.

As used herein, term " immunogenic composition " or " immunogenic formulation ", which refer to work as, is applied to vertebrate, Especially animal (such as mammal) when, will induce immune response (including Th1 immune response) preparation.

Terms used herein " indicant " refer to the expression of result or result, including technical staff can assess and/or Determine any information of a possibility that whether subject suffers from specified disease or risk, quantity, ratio, signal, symbol, label or Annotation.In the present case, " indicant " is optionally used together with other Clinical symptoms, to realize subject Th1 The determination of immune state.This indicant is " determination ", is not meant to imply that indicant is 100% accurate.Skilled Indicant can be used together to realize diagnosis by clinician with other clinical markers.

" connector " refers to the molecule or molecular group (such as monomer or polymer) of two molecules of connection, and being commonly used in will Two molecules are placed in required configuration.In specific embodiments, " peptide linker " refer to connection two albumen, polypeptide, peptide, The amino acid sequence of structural domain, area or motif, and can provide and two sub-combination (for example, oligomerization) structural domain phase interactions With compatible interval function, so that gained polypeptide and target molecules keep specific binding affinity or keep signaling activity (for example, PD-L2 polypeptide).In certain embodiments, connector includes about 2 to about 35 amino acid, for example, or about 4 to about 20 A amino acid or about 8 to about 15 amino acid or about 15 to about 25 amino acid.

As used herein, term " part " refers to a part of molecule, can be the functional group of intramolecular, one group of function The specific group of group and/or atom, is responsible for the Characteristic chemical, biology and/or medicinal characteristic of molecule.

" acquisition " means to occupy.The sample so obtained includes such as separation or the nucleic acid extraction derived from particular source Object or polypeptide extract.For example, extract can be separated directly from subject's biofluid or tissue.

" oligomerization domain " used herein refers to protein structure domain, directly or by bridging molecule preferentially with one Or the interaction of multiple other protein structure domains or combine, wherein the interaction of other protein structure domains substantially contribute to or Oligomerization is effectively facilitated (that is, forming dimer, tripolymer, the tetramer, pentamer, six aggressiveness, heptamer, eight aggressiveness, nine poly- Body, ten aggressiveness, 11 aggressiveness, ten dimers or more advanced oligomer, its can be same oligomer or different oligomer).It is this " complementary " oligomerization domain includes dimerization domain (for example, immunoglobulin Fc domain, leucine zipper etc.), three Multimerisation domain (for example, the catalytic subunit of Escherichia coli aspartate carbamyl-transferase (ATCase), from T4 bacteriophage it is secondary " foldon " the trimerization sequence of fibrin (fibritin), neck region peptide, people's Curosurf protein D, oligomerization crimp spiral shell Revolve adhesin, complementary heptad repeat region of I class enveloped virus fusion protein etc.), tetramerization structural domain is (for example, the viscous connection egg of four arms White coiled-coil domain), five multimerisation domains are (for example, the five of tryptophan zipper or cartilage oligo-substrate protein (COMP) Multimerisation domain etc.) and six multimerisation domains (for example, tail portion from IgA antibody heavy chain C-terminal), when needing, it can combine Using to form more advanced oligomer, such as heptamer, eight aggressiveness, nine aggressiveness, ten aggressiveness, 11 aggressiveness, ten dimers.

Term " being operably connected " as used herein or " being operatively connected " refer to juxtaposition, wherein at the component In the relationship for allowing them to work in a manner of its expection.For example, with interest nucleotide sequence (for example, coding and/or non-volume Code sequence) the adjusting sequence (for example, promoter) of " being operably connected " refers to that control sequence is relative to interest nucleotide sequence Position and/or positioning, permission the sequence is expressed under conditions of compatible with control sequence.Control sequence does not need and interest core Nucleotide sequence is adjacent, as long as they play the role of that it is instructed to express.Thus, for example between promoter and coded sequence There may be the non-coding sequence of insertion (for example, untranslated but sequences of transcription), and promoter sequence still can consider It " is operably connected " with coded sequence.Equally, PD-L2 polypeptide " operationally connects at least one hetero-oligomerization structural domain Connect " it include position and/or positioning of the oligomerization domain relative to PD-L2 polypeptide, to allow PD-L2- oligomerization domain embedding The self assembly of polypeptide is closed to form polypeptide complex.

Term " patient ", " subject ", " host " or " individual " interchangeably used herein refers to any subject, special It is not vertebrate subject, or even more specifically mammalian subject, needs to treat or prevent.Fall into model of the present invention Enclose any member that interior suitable vertebrate includes but is not limited to notochord subgenus, including primate (for example, people, monkey and Ape, and the monkey species including coming from Macaca (Macaca) (for example, machin, such as macaque (Macaca fascicularis) And/or rhesus macaque (Macaca mulatta)) and baboon (Papio ursinus) and marmoset (come from marmoset category (Callithrix) species), the Squirrel monkey species of (come from Saimiri (Saimiri)) and golden monkey be (from Chinese tamarisk Lagothrix (Saguinus) species) and the species (such as chimpanzee (Pan troglodytes)) of ape, rodent (for example, small Mouse, rat, cavy), Lagomorpha (for example, rabbit, hare), Bovidae (for example, ox), sheep section (for example, sheep), goat section (for example, goat), Suidae (for example, pig), equine (for example, horse), Canidae (for example, dog), cat family (for example, cat), fowl (for example, Chicken, turkey, duck, goose, companion bird (such as canary, budgerigar)), marine mammal (for example, dolphin, whale), creep it is dynamic Object (snake, the frog, lizard etc.) and fish.Preferred subject is to need to cause to enveloped virus fusion protein or fusion protein compound Immune response people.However, it is to be understood that above-mentioned term is not meant to that there are symptoms.

As used interchangeably herein, " PD-L2 activity ", " bioactivity of PD-L2 " or " functional activity of PD-L2 " are Refer to, measured in vivo or in vitro according to standard technique, PD-L2 polypeptide or nucleic acid molecules to PD-L2 respond cell or tissue or The activity that PD-L2 polypeptide binding partners are played.In some embodiments, PD-L2 activity is direct activity, such as with The combination of PD-L2 binding partners.As used herein, " target molecules " or " binding partners " are that PD-L2 polypeptide is naturally tied The molecule for closing or interacting, to realize the function that PD-L2 is mediated.In specific embodiments, PD-L2 target molecules are selected Molecule b (RGMb) is instructed from PD-1 receptor and repellency.Alternatively, PD-L2 activity is indirect activity, for example, by PD-L2 polypeptide with The cell signaling activity that natural binding partner (for example, PD-1 or RGMb) interaction mediates.This document describes PD-L2 Biological activity.For example, PD-L2 polypeptide of the invention can have one or more following activity: 1) combine and/or adjust by The activity of body PD-1 or other PD-L2 natural binding partners (such as RGMb), 2) conduction of intracellular or intercellular signal is adjusted, 3) it adjusts the activation of immunocyte, such as T lymphocyte and 4) adjusts biology (such as mammal, such as people or other primates Animal) immune response, including Th1 immune response.

Term " PD-L2 expression " and " PD-L1 expression " respectively refer to the transcription and/or translation and/or work of PD-L2 and PD-L1 Property.Several method can be used to determine the level of PD-L2 and PD-L1 expression, such as described herein.

" PD-L2 polypeptide " refers to the polypeptide with the amino acid sequence corresponding to PD-L2 molecule.The term includes but unlimited In the polypeptide with amino acid sequence, wherein any one institute in the amino acid sequence and SEQ ID NO:1,2,3,4,5 or 6 The sequence stated have at least 70% (at least 71% at least 99% and between all integer percents) sequence identity or Similitude.It further includes the natural allelic variation of PD-L2 polypeptide, and the natural allelic variation may exist and occur In a biology into another biology.In addition, glycosylated degree and position or other posttranslational modifications, it can be according to selected The property of the host and host cell environment that select and change.Term " PD-L2 polypeptide " also aims to the PD-L2 including its precursor forms Polypeptide, and it is processed to generate those of their own biologically active form.Its further comprise relative to reference or Naturally occurring PD-L2 polypeptide and PD-L2 polypeptide Jing Guo chemical modification, and/or relative to reference or naturally occurring PD-L2 The PD-L2 polypeptide that changes for polypeptide containing one or more amino acid sequences, and/or relative to reference or naturally occurring complete PD-L2 polypeptide (including PD-L2 signal for long or precursor PD-L2 polypeptide or its structural domain comprising truncated amino acid sequence Peptide, IgV and IgC structural domain, extracellular domain, transmembrane domain and cytoplasmic domains).Alternatively, or in addition, PD-L2 polypeptide (including its Compound) can show relative to reference or naturally occurring PD-L2 polypeptide different characteristics, including change (for example, mentioning It is high) stability and (for example, enhancing) bioactivity for changing, such as, but not limited to: 1) enhancing and receptor PD-1 or other The combination and/or signal transduction of PD-L2 natural binding partner (such as RGMb), 2) the intracellular or intercellular signal of enhancing passes Lead, 3) activated immune cell of enhancing, such as T lymphocyte and 4) in subject (such as mammal, as people or its His primate) enhancing immune response (including Th1 immune response).Term " PD-L2 polypeptide " further includes having slightly to repair The protein molecular of the amino acid sequence of decorations, such as the end N- (lack or add including -terminal amino acid) with modification are more Peptide, and/or relative to reference or naturally occurring PD-L2 polypeptide and through the polypeptide of chemical modification.PD-L2 polypeptide further includes comparing The protein molecular of substantially the same or more preferable bioactivity is shown in reference or naturally occurring PD-L2 polypeptide, or including phase The protein molecular of bioactivity substantially change or reduced is shown for reference or naturally occurring PD-L2 polypeptide.

" pharmaceutically acceptable carrier " refers to solid or liquid filler, diluent or encapsulating substance, can be safely For being locally or systemically applied to animal, preferably mammal, including people.Representative pharmaceutically acceptable carrier include with Under it is any or whole: it is solvent known to persons of ordinary skill in the art, decentralized medium, coating, surfactant, anti-oxidant Agent, preservative (for example, antibacterial agent, antifungal agent), isotonic agent, absorption delaying agent, salt, preservative, drug, drug stabilizing agent, Gel, adhesive, excipient, disintegrating agent, lubricant, sweetener, flavoring agent, dyestuff, similar substance and combinations thereof are (referring to example Such as Remington ' s Pharmaceutical Sciences, the 18th edition, Mack Printing Company, 1990, Pp.1289-1329 is incorporated herein by reference).Unless any routine carrier is incompatible with active constituent, its use is otherwise considered In pharmaceutical composition.

Terms used herein " polynucleotides " or " nucleic acid " indicate mRNA, RNA, cRNA, cDNA or DNA.The term is logical The nucleotide for referring to the polymerized form that length is at least ten base can be ribonucleotide or deoxynucleotide or appoint The modified forms of one type Nucleotide.The term includes single-stranded and double-stranded form DNA.

" polypeptide ", " peptide ", " albumen " and " protein molecular " is used interchangeably herein, is referred to and is polymerize comprising amino acid residue Object or the molecule being made of amino acid residue polymer, and it is related to its variant and synthetic analogues.Therefore, these terms are applicable in In such amino acid polymer, wherein one or more amino acid residues are the non-naturally occurring amino acid of synthesis, such as The chemical analog of corresponding naturally occurring amino acid and naturally occurring amino acid polymer.

Terms used herein " recombination of polynucleotide " refer to by by nucleic-acid manipulation in nature there is usually no Form and the polynucleotides formed in vitro.For example, recombination of polynucleotide can be the form of expression vector.In general, such table It include that the transcription and translation being operably connected with nucleotide sequence adjusts nucleic acid up to carrier.

" recombinant polypeptide " refers to the polypeptide using recombinant technique (i.e. by expression recombination of polynucleotide) preparation.

" regulating element ", " adjusting sequence ", " control element ", " control sequence " etc. are used interchangeably herein, to refer to Positioned at upstream of coding sequence (5' non-coding sequence), internal or downstream (3' non-coding sequence) nucleotide sequence, directly or Affect indirectly transcription, RNA processing or the stability or translation of related coding sequences.Regulating element includes enhancer, starting Son, translation leader, introne, Rep recognition component, intergenic region and polyadenylation signal sequence.They include Natural and composition sequence and the sequence that can be synthesis and native sequences combination.

Terms used herein " sample " include times that can be extracted from subject, is untreated, handling, diluting or being concentrated What biological sample.Sample may include but be not limited to biofluid, for example, whole blood, serum, red blood cell, leucocyte, blood plasma, saliva, Urine, excrement (i.e. excreta), tear, sweat, sebum, nipple aspirate, ductal lavage fluid, tumour exudate, synovia, ascites, abdomen Film liquid, amniotic fluid, cerebrospinal fluid, lymph, fine needle aspiration object, amniotic fluid, any other body fluid, cell pyrolysis liquid, cell secretory product, Inflammation liquid, sperm and vaginal fluid.Sample may include tissue sample and biopsy, tissue homogenate etc..Expedient sample can Sample including the biomarker containing any one or more of detectable amount teaching herein.Suitably, sample can pass through Invasive methods are readily available, which allows that sample is removed or separated from subject.In certain embodiments, sample Product contain blood, especially peripheral blood or part thereof or extract.In general, sample includes haemocyte, such as maturation, prematurity Or developmental leucocyte, including lymphocyte, polymorphonuclear leukocyte, neutrophil leucocyte, monocyte, granulophilocyte, Basophilic granulocyte, coelomocyte, haemocyte, eosinophil, megacaryocyte, macrophage, dendritic cells, natural kill The part (for example, nucleic acid or protein part) of cell or this cell.In specific embodiments, sample includes leucocyte, Including peripheral blood mononuclear cells (PBMC).

" self assembly " refers to the process of spontaneous assembling higher structure, depends on the component (for example, molecule) of higher structure Mutual nature attraction.Based on size, shape, composition or chemical property, it usually passes through random motion and the key of molecule It is formed and is occurred.

Term " sequence identity " as used herein refers in comparison window in nucleotide-nucleotide or amino acid- On the basis of amino acid, the identical degree of sequence.Therefore, " percentage of sequence identity " calculates as follows: in comparison window Compare two optimal comparison sequences, determine occur identical nucleic acid base (such as A, T, C, G, I) or identical amino in two sequences Sour residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) position number to generate the number of matching position, by the number of matching position divided by comparison window In total number of positions (that is, window size) and result is generated to the percentage of sequence identity multiplied by 100.The present invention considers Overall length IL-22 polypeptide and its bioactive fragment are used in method and system of the invention.In general, overall length IL-22 polypeptide Bioactive fragment may participate in interaction, such as intramolecular or intermolecular interaction.

" similitude " refers to the percentage number of amino acid that is identical or constituting conservative substitution, as determined in table 1 above and 2 Justice.Use sequence comparison program such as GAP (Deveraux et al. 1984, Nucleic Acids Research 12:387- 395) similitude is determined.In this way, there is the sequence of similar-length or substantially different length with herein cited sequence, It can be compared by being inserted into vacancy in comparison, such as this vacancy is determined by comparison algorithm that GAP is used.

For describing the term of sequence relation between two or more polynucleotides or polypeptide, including " reference sequences ", " comparison window ", " sequence identity ", " percentage of sequence identity " and " basic identity ".The length of " reference sequences " is At least 12, but usually 15 to 18, typically at least 25 monomeric units (including nucleotide and amino acid residue).Because two A polynucleotides can respectively contain between (1) two polynucleotides similar sequence (that is, only one of complete polynucleotide sequence Point), the different sequence between (2) two polynucleotides, the sequence between two (or more) polynucleotides is more usual It is carried out by comparing the sequence of two polynucleotides in " comparison window ", to identify and compare the partial zones of sequence similarity Domain." comparison window " refers to the conceptual segment of at least six continuous position, typically about 50 to about 100, more typically about 100 To about 150, wherein after two sequence optimal comparisons, sequence is compared with the reference sequences with identical continuous position number. For the optimal comparison of two sequences, compared with reference sequences (it is without addition or missing), comparison window may include about 20% or less addition or missing (that is, vacancy).For comparing the sequence optimal comparison of comparison window, computer can be passed through The algorithm of change implement (GAP, BESTFIT in Wisconsin Genetics Software Package Release 7.0, FASTA and TFASTA, Genetics Computer Group, 575Science Drive Madison, WI, USA) or pass through Inspection and the optimal comparison generated by selected any various methods in comparison window (that is, generate highest percentage Homology) Lai Shixian.Can also with reference to BLAST series of programs, such as Altschul et al., 1997, Nucl.Acids Disclosed in Res.25:3389.Being discussed in detail for sequence analysis can be in Ausubel et al. " Current Protocols in It is found in the 15th chapter of unit 19.3 of Molecular Biology ", John Wiley&Sons Inc, 1994-1998.

As used herein, term " single-stranded " is the single linear for the amino acid being covalently attached and continuously arranges.

As used herein, term " solubility " polypeptide (such as solubility PD-L2 polypeptide) refers to non-naturally occurring polypeptide, Usually film combines, and works under non-film bonding state now, while remaining binding molecule and being combined pair by its film The ability for answering object (such as PD-1) to be identified.

" Th1 related disease " or " Th1 associated disease " is interchangeably used herein, refers to the development with Th1 immune response Relevant disease." Th1 immune response " used herein refers to the proliferation or increased differentiation of Th1 cell.Suitably, by with Lower identification Th1 related disease or illness: (1) level of Th1 cell, Th1 cell factor and/or Th1 antibody is more than usually to exist The level found in people, animal or cell culture；(2) pathology discovery relevant to disease or medical conditions, by applying Simulation can be tested in animal with the medicament of up-regulation Th1 cell Proliferation or differentiation；Or (3) pass through with inhibit Th1 cell Proliferation or The medicament of differentiation is handled, and the pathology induced in the experimental animal model of disease or medical conditions can be suppressed or eliminated It learns.In the relevant disease of most of Th1, at least meet two in three conditions.

As used herein, term " treatment ", " processing " etc. refer to the pharmacology and/or physiological role needed for obtaining.Just Completely or partially for prevention disease or its symptom, which can be preventative, and/or just partially or completely cure disease And/or be attributable to for the ill-effect of disease, it can be therapeutic.As used herein, " treatment " covers mammal Any treatment of (especially people) disease, and include: that (a) prevention disease generation may be susceptible to suffer from the disease but not yet be diagnosed as In subject with the disease；(b) inhibit disease, that is, prevent its development；And (c) alleviate disease, that is, cause disappearing for disease It moves back.

" carrier " refers to polynucleotide molecule, suitably the DNA for example derived from plasmid, bacteriophage, yeast or virus points Son can be inserted into or clone thereto polynucleotides.Carrier can be containing one or more unique restriction sites, and are limiting It can independently be replicated in fixed host cell (including target cell or tissue or progenitor cells or its tissue), or can be with restriction Host genome integration so that the sequence of clone can be bred.Therefore, carrier can be autonomously replicationg vector, i.e., as dyeing Carrier existing for external entity, is replicated independently of chromosome replication, such as the outer member of linear or closed hoop plasmid, chromosome Part, minichromosome or artificial chromosome.Carrier may include any method for ensuring self-replacation.Alternatively, carrier can be Such carrier is integrated into the chromosome in genome and being integrated with and replicates together when it is introduced into host cell.Carrier System may include single carrier or plasmid, two or more carriers or plasmid, they contain host cell base to be introduced together Because of the total DNA or transposons of group.The selection of carrier generally depends on the compatibility of carrier with the host cell that import carrier.? In the case where the present invention, carrier is preferably carrier derived from virus or virus, can in animal and preferred mammal cell Operatively play a role.This carrier can be derived from poxvirus, adenovirus or yeast.Carrier can also include selected marker Object, such as antibiotics resistance gene can be used for selecting suitable transformant.The example of such resistant gene is this field skill Known to art personnel, and including assigning antibiotic kanamycins and G418The nptII gene of resistance and tax Give the hph gene of antibiotic hygromycin B resistance.

Term " wild type ", " natural " and " naturally occurring " are used interchangeably herein, and referring to ought be from naturally depositing Source separate when gene or gene product with the gene or gene product feature.It is wild type, natural or naturally occurring Gene or gene product (for example, polypeptide) be most commonly observed in group, therefore arbitrarily devised gene or gene product " normal " or " wild type " form.

Unless expressly stated otherwise, otherwise each embodiment as described herein is applied after making necessary modification in detail In each embodiment.

2. polypeptide complex

Surprisingly, it was found that the PD-L2 in IEC- interaction cell (such as APC (for example, dendritic cells)) Expression is inversely proportional with the severity of Th1 associated disease, and PD-L2 is that establish Th1 immune necessary.In other words, if PD-L2 expression on APC is lower than with there are the immune-related threshold value of normal or undamaged Th1, then Th1 immunity is impaired.This Inventor also found that the aggregation of PD-L2 can inhibit the combination of PD-L1 and PD-1 on the surface APC, so that PD-L1 be inhibited to fight The immune suppression function of original specificity IEC (such as T cells with antigenic specificity).This makes inventor compare the difference of PD-L2 Effect of the oligomer to blocking PD-L1 in conjunction with PD1.It is worth noting that, the dimeric forms of discovery PD-L2 cannot effectively hinder Break this combination.However, the present inventors have additionally discovered that more advanced PD-L2 oligomer, that is, have 3 or higher oligomerization degree (for example, 3,4,5,6,7,8,9,10,11,12 etc.), have than dimer PD-L2 in conjunction with PD-1 higher affinity, and it is this more PD-L1 can be significantly reduced to the inhibiting effect of IEC function, including CD4 in advanced PD-L2 oligomer⁺T cell function.Therefore, The present invention provides the PD-L2 oligomer with 3 or higher oligomerization degree (for example, 3,4,5,6,7,8,9,10,11,12 etc.), uses It is immunized in adjusting Th1 and treats Th1 related disease, as described in more detail below.

Therefore, the present invention provides can be used for stimulating or enhance the immune polypeptide complex of Th1, the compound is usually by formula (I) shown in:

[P]_n (I)

Wherein:

P, independently represents protein molecular when occurring every time, the protein molecular includes PD-L2 polypeptide, by PD-L2 polypeptide Composition is substantially made of PD-L2 polypeptide；And

N represents the integer greater than 2.

2.1 PD-L2 polypeptides

PD-L2 is transmembrane protein, and is in monomer form, and it includes signal peptides, IgV and IgC structural domain, is constituted The extracellular domain (also referred herein as " extracellular domain ") of molecule, IgV spline structure domain completely or partially responsible PD-1 combine with And other function (including signal transduction).PD-L2 also contains short intracytoplasmic domain and single transmembrane domain.

The non-limiting example of PD-L2 albumen can be selected from following PD-L2 ortholog thing:

People PD-L2

Chimpanzee PD-L2

Mouse PD-L2

A kind of polypeptide, with sequence shown in SEQ ID NO:1 to any of 3 have at least 70% (and at least 71% to 99% and all integer percents therebetween) sequence similarity or identity.

Useful PD-L2 polypeptide includes soluble fragments, is the appropriate segment of PD-L2, can be from generation cell It falls off, secrete or otherwise extracts.In some embodiments, PD-L2 polypeptide includes the entire extracellular domain of PD-L2.PD- The extracellular domain of L2 includes mammal PD-L2 about 20 to the about amino acid of amino acid 221 or its active fragment.In other implementations In scheme, PD-L2 polypeptide includes IgC the and IgV structural domain of PD-L2.In other embodiments, PD-L2 polypeptide includes PD-L2 IgV structural domain.

In specific embodiments, PD-L2 polypeptide includes PD-L2 extracellular domain, is made of PD-L2 extracellular domain or substantially It is made of PD-L2 extracellular domain, illustrated examples include:

People's PD-L2 extracellular domain plus signal peptide

Chimpanzee PD-L2 extracellular domain plus signal peptide

Mouse PD-L2 extracellular domain plus signal peptide

People's PD-L2 extracellular domain

Chimpanzee PD-L2 extracellular domain

Mouse PD-L2 extracellular domain

A kind of extracellular domain has at least 70% (and at least 71% with sequence shown in SEQ ID NO:4 to any of 9 To 99% and all integer percents therebetween) sequence similarity or identity.

In some embodiments, PD-L2 polypeptide includes at least part of PD-L2 transmembrane domain.

Many other mammal PD-L2 sequences (including primate sequence) are known in the art, such as Disclosed in GenPept database or Onlamoon et al. (Immunology 124:277-293,2008).

It can make protein molecular oligomerization of the invention by any suitable method.For example, oligomer can passing through Be connected between protein molecular and formed, such as by using heterobifunctional linker or passes through oligomerization domain and albumen point The PD-L2 polypeptide of son is operatively connected.

2.2 isodigeranyl function connects reagents

Protein molecular and other protein moleculars are connected with generate polypeptide complex of the invention can be it is direct or It connects.For example, heterobifunctional linker can be realized by being connected chemically or be passed through to the connection of two or more protein moleculars Promoted.It is for the formation covalent bond between amino and sulfydryl and by many heterobifunctional crosslinkers that sulfydryl introduces albumen It is well known by persons skilled in the art (see, e.g. PIERCE CATALOG, ImmunoTechnology Catalog& Handbook, 1992-1993, which depict making and using for these reagents, and provide commercial source for these reagents；Also See, e.g. Cumber et al. Bioconjugate Chem.3:397-401,1992；Thorpe et al. Cancer Res.47: 5924-5931,1987；Gordon et al. Proc.Natl.Acad Sci.USA 84:308-312,1987；Walden et al. J.Mol.Cell Immunol.2:191-197,1986；Carlsson et al. Biochem.J.173:723-737,1978； Mahan et al. Anal.Biochem.162:163-170,1987；Wawrzynczak et al. Br.J.Cancer 66:361-366, 1992；Fattom et al. Infection&Immun.60:584-589,1992).These reagents can be used in PD-L2 polypeptide and another Covalent bond is formed between a kind of PD-L2 polypeptide, including but not limited to: N- succinimido -3- (2- pyridyl group two is thio) third Acid esters (SPDP；Disulfide bond connector)；Sulfosuccinimide base 6- [3- (2- pyridyl group two is thio) propionamido-] capronate (sulphur Base-LC-SPDP)；Succinimide Epoxide carbonyl-α-methylbenzyl thiosulfates (SMBT, the di-sulfate connector being obstructed)； Succinimido 6- [3- (2- pyridyl group two is thio) propionamido-] capronate (LC-SPDP)；Sulfosuccinimide base 4- (N- maleimidomehyl) hexamethylene -1- carboxylate (sulfo group-SMCC)；Succinimido 3- (2- pyridyl group two is thio) fourth Acid esters (SPDB；The disulfide bond connector being obstructed)；Sulfosuccinimide base 2- (7- azido -4- methylcoumarin -3- acetyl Amine) ethyl -1,3'- dithiopropionic acid ester (SAED)；Sulfosuccinimide base 7- azido -4- methylcoumarin -3- acetic acid Ester (SAMCA)；Sulfosuccinimide base -6- [Alpha-Methyl-α-(2- pyridyl group) toluamide] capronate (sulfo group-LC- SMPT)；1,4- bis--[3'- (2'- pyridyl group two is thio) propionamido-] butane (DPDPB)；4- succinimido oxygroup carbonyl Base-Alpha-Methyl-α-(2- pyridylthio)-toluene (SMPT, the di-sulfate connector being obstructed)；Sulfosuccinimide base -6- [Alpha-Methyl-α-(2- pyrimidine radicals-thio) toluamide] capronate (sulfo group-LC-SMPT)；M- maleimidobenzoyl Base-N- hydroxy-succinimide ester (MBS)；M- maleimidobenzoyl-N- weight ratio-succinimide ester (sulfo group-MBS)；N- succinimido (4- iodoacetyl) Aminobenzoate (SIAB；Thioether linker)；Sulfosuccinic acyl is sub- Amido-(4- iodoacetyl) Aminobenzoate (sulfo group-SIAB)；Succinimido -4- is (to dimaleoyl imino-hexichol Base) butyrate (SMPB)；Sulfosuccinimide base -4- (to maleimido-phenyl) butyrate (sulfo group-SMPB)；It is folded Nitrogen base benzoyl hydrazine (ABH).For example, these connectors can be applied in combination with peptide linker, such as those increase flexibility or dissolution Degree or the peptide linker for providing or eliminating steric hindrance.It can be used well known by persons skilled in the art for connecting peptide molecule To any other connector of another molecule.General aspects make in conjunction with gained molecule and PD-1 or another kind target molecules (for example, Another homoreceptor).

2.3 oligomerization domain

The interaction of three or more PD-L2 polypeptides can be directly or indirectly connected to energy itself by them It enough interacts to form any part of rock-steady structure or other polypeptides and is promoted.For example, can be connected by oligomerization Individual PD-L2 polypeptide chain is to form protein molecular of the invention, and thus the oligomerization of polypeptide is mediated by oligomerization domain. In general, oligomerization domain is provided at least 3,4,5,6,7,8,9,10,11,12 or the albumen of even more polypeptides containing PD-L2 Stable protein-protein interaction is formed between molecule.The oligomerization domain of individual PD-L2 polypeptide can be with the present invention The oligomerization domain of another PD-L2 polypeptide in polypeptide complex is different, as long as different oligomerization domains is " complementary ", they just preferentially interact with each other or be combined with each other with allow protein molecular oligomerization (form oligomer, As tripolymer, the tetramer, pentamer, six aggressiveness, heptamer, eight aggressiveness, nine aggressiveness, ten aggressiveness, 11 aggressiveness, ten dimers or More advanced oligomer can be same oligomer or different oligomer).

In general, oligomerization domain includes any amino acid sequence for being capable of forming stable protein-protein interaction. Oligomerization domain can be interacted by immunoglobulin sequences (for example, Fc structural domain；See for example, international patent publications 2005/063816 US of WO 93/10151 and WO；U.S. Patent Publication No. 2006/0024298；U.S. Patent number 5,457, 035), leucine zipper is (for example, from Nuclear transformation albumen fos and jun or c myc or come from General Control of Nitrogen (GCN4)), hydrophobic region, hydrophilic area or formed between homologous or hetero-oligomer chimeric molecule The free sulfhydryl groups of intermolecular disulfide bond.In addition, oligomerization domain may include the amino acid sequence containing protrusion, the protrusion with Amino acid sequence comprising hole is complementary, such as is described in U.S. Patent number 5,731,168；International Patent Publication No. WO 98/ 50431 and WO 2005/063816；Ridgway et al. (1996) Protein Engineering 9:617-621.It can be with engineering This oligomerization region is transformed, so that steric interaction not only promotes stable interaction, but also is compared with homodimer, More promote to form heterodimer from chimeric monomer mixture.In general, passing through with biggish side chain (for example, tyrosine or color ammonia Acid) replace the small amino acid side chains at the first polypeptide interface, to construct protrusion.Optionally, by with lesser amino acid side chain (example Such as, alanine or threonine) the relatively big amino acid side chains that replace the second polypeptide interface, generate have with protrusion it is same or similar big Small complimentary cavity.Exemplary oligomerization domain is as described below.

Anywhere PD-L2 polypeptide, such as any provided herein, can be attached, but usually by its N- or The end C- is connected to the end N- or C- of the oligomerization module comprising at least one oligomerization domain, to form chimeric polyeptides. Connection can carry out indirectly directly or by connector.In addition, chimeric polyeptides can be fusion protein or can be by being connected chemically It is formed, such as by covalently or non-covalently interacting.For example, when preparation includes the widow at least one oligomerization domain When the chimeric polyeptides of dimerization module, the nucleic acid for encoding PD-L2 polypeptide can be directly or indirectly or optionally by joint peptide and volume The nucleic acid of code oligomerization module is operably connected, to form nucleic acid construct.In general, construct encoding chimera polypeptide, wherein The end C- of PD-L2 polypeptide is connect with the end N- of oligomerization module.In some cases, construct codified chimeric polyeptides, Wherein the end N- of PD-L2 polypeptide and the end C- of oligomerization domain connect.

For example, the embodiment that at least one of them oligomerization domain is operatively coupled on PD-L2 polypeptide downstream In, the protein molecular forms, comprising single polypeptide chain, the single polypeptide chain shown in formula (II) shown in formula (II) substantially by formula (II) composition of single polypeptide chain shown in:

PD-L2-L-OMD_A (II)

Wherein:

PD-L2 represents PD-L2 polypeptide；

OMD_AIt is oligomerization domain, forms i subunit OMD_AOligomer (OMD_A)_i, wherein i >=3, suitably for 3,4,5 or 6；And

L is key or peptide linker.

Alternatively, the protein molecular includes single polypeptide chain, the single polypeptide chain group as shown in formula (III) shown in formula (III) It is formed at, the substantially single polypeptide chain shown in formula (III):

PD-L2-L-OMD_A-L-OMD_B (III)

Wherein:

OMD_AIt is oligomerization domain, forms i subunit OMD_AOligomer (OMD_A)_i, wherein i >=2, suitably for 2,3,4,5 or 6；

L independently represents key or peptide linker when occurring every time；And

OMD_BIt is oligomerization domain, forms j subunit OMD_BOligomer (OMD_B)_j, wherein j is greater than the whole of i Number, is suitably i+1, i+2, i+3, i+4, i+5 or i+6；

At least one of them oligomerization domain is operatively coupled in the embodiment of PD-L2 polypeptide upstream, egg White molecule forms, comprising single polypeptide chain, the single polypeptide chain shown in formula (IV) shown in formula (IV) substantially by shown in formula (IV) Single polypeptide chain composition:

OMD_A-L-PD-L2 (IV)

Wherein:

OMD_AIt is oligomerization domain, forms i subunit OMD_AOligomer (OMD_A)_i, wherein i >=3, suitably for 3,4,5 or 6；

L is key or peptide linker；And

PD-L2 represents PD-L2 polypeptide.

Alternatively, the protein molecular is formed comprising single polypeptide chain, the single polypeptide chain shown in formula (V) shown in formula (V), base Single polypeptide chain shown in formula (V) forms on this:

OMD_B-L-OMD_A-L-PD-L2 (V)

Wherein:

OMD_BIt is oligomerization domain, forms j subunit OMD_BOligomer (OMD_B)_j, wherein j >=2, suitably for 2,3,4,5 or 6；

L independently represents key or peptide linker when occurring every time；And

OMD_AIt is oligomerization domain, forms i subunit OMD_AOligomer (OMD_A)_i, wherein i is greater than the whole of j Number, is suitably j+1, j+2, j+3, j+4, j+5 or j+6；And

PD-L2 represents PD-L2 polypeptide.

Many oligomerization domains be it is known in the art, representative example includes:

2.3.1 immunoglobulin domains

Oligomerization domain includes comprising those of free sulfhydryl groups part, and wherein free sulfhydryl groups part can be with other amino The oligomerization domain of acid sequence reacts to form intermolecular disulfide bond.For example, oligomerization domain may include immunoglobulin point A part of son, such as a part from IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgM or IgE.In general, this part It is constant region for immunoglobulin (Fc).The preparation that fusion protein has been described contains and is fused to antibody-derived polypeptides difference The partially polypeptide (including Fc structural domain), see, for example, Ashkenazi et al. Proc.Natl.Acad.Sci USA 88: 10535,1991；Byrn et al. Nature, 344:667,1990；And Hollenbaugh and Aruffo (2002) are in Current " Construction of Immunoglobulin Fusion Proteins " in Protocols in Immunology Ten chapter pp.10.19.1-10.19.11.

In some embodiments, oligomerization domain includes full-length immunoglobulin polypeptide.Alternatively, immunoglobulin is more Peptide is less than overall length, that is, contains heavy chain, light chain, Fab, Fab₂, Fv or Fc.In one example, PD-L2 polypeptide-immunoglobulin Chimeric polyeptides are assembled into exclusive OR with-oligomer, especially as the tetramer.It can use the chain or basic with different structure Unit assembles exclusive OR with-oligomer.For example, PD-L2 polypeptide can be fused to all or part of immunoglobulin molecules, All or part of C including immunoglobulin molecules_H、C_L、V_HOr V_LStructural domain (see, for example, U.S. Patent number 5,116,964). Conversion has the mammalian cell of Suitable nucleic acid molecules that can easily generate and secrete chimeric PD-L2 polypeptide-immune globulin White polypeptide.Secreted form includes that PD-L2 polypeptide those of is present in heavy chain and the different tetramer of light chain, wherein PD-L2 polypeptide with One or more light chain or heavy chain fusion are included therein up to four and all four variable region analogs substituted different four Aggressiveness.In some instances, a kind of or more than one nucleic acid fusion molecule can be transformed into host cell to generate oligomerization Body, wherein the PD-L2 polypeptide portion of oligomer is identical or different.

Fc structural domain

In general, the immunoglobulin part of PD-L2 polypeptide chimeric polyeptides includes the heavy chain of immunoglobulin polypeptides, it is most common Be heavy chain constant domain.The heavy chain constant region sequence of exemplary human IgG hypotype is selected from following sequence:

Wherein C_H1Structural domain corresponds to amino acid 1-98, and hinge area corresponds to amino acid 99-110, C_H2Structural domain corresponds to Amino acid 1 11-223 and C_H3Structural domain corresponds to amino acid 224-330.

In one example, immunoglobulin polypeptides chimeric protein may include the area Fc of immunoglobulin polypeptides.In general, this Kind fusion at least retains the functional activity hinge of immunoglobulin heavy chain constant region, C_H2And C_H3Structural domain.For example, IgG1's is complete Long Fc sequence includes the amino acid 99-330 of sequence shown in SEQ ID NO:10.The Fc sequence of exemplary human IgG1 is:

It contains C shown in almost all of hinge sequence and SEQ ID NO:10_H2And C_H3The complete sequence of structural domain Column.

Alternatively, the Fc polypeptide of human IgG1 is selected from:

Other than human IgG1 Fc, other areas Fc also may include embedding in PD-L2 polypeptide-oligomerization module provided herein In hop protein.For example, having feelings to be minimized by the immunological effect function that Fc/Fc γ receptor (Fc/Fc γ R) interaction mediates Under condition, it may be considered that merged with the IgG isotype for being difficult to raise complement or immune effector cell, such as the Fc of IgG2 or IgG4. In addition, Fc fusion can contain immunoglobulin sequences, the substantially immunoglobulin gene by belonging to any antibody isotype Coding, including but not limited to IgG (including people's subclass IgG1, IgG2, IgG3 or IgG4), IgA (including people's subclass IgA1 and IgA2), IgD, IgE and IgM class antibody.In addition, connector can be used for for Fc connecting with another polypeptid covalence to generate Fc chimera.

Consider that the Fc structural domain of modification is used for the chimera with PD-L2 polypeptide herein.In some instances, the area Fc is modified So that its show and FcR change combination, so as to cause than the area wild-type immunoglobulin heavy chain Fc effector function (that is, More or less) the effector function changed.Therefore, the Fc structural domain of modification can have the affinity of change, including but not limited to The affinity of Fc receptor is increased or decreased or do not had.For example, different IgG subclass has different affinity to Fc γ R, Middle IgG1 and IgG3 usually essentially better than IgG2 and IgG4 bind receptor.The Fc structural domain of modification is those skilled in the art It known to member, and is described in the literature, see, e.g. U.S. Patent number 5,457,035；U.S patent publication No. US 2006/0024298；Example sex modification is used for International Patent Publication No. WO 2005/063816.

2.3.2 coiled-coil domain

A kind of common structure motif for participating in protein oligomerization is coiled-coil domain, and this structural domain can also be made PD-L2 polypeptide complex is used to prepare for oligomerization domain.α-helixstructure motif of curling itself can form spiral, and And two, three, four or five alpha-helixes can surround each other and form left-handed supercoil, referred to as " coiled coil ", even if So designed artificial dextrorotation supercoil (Burkhard et al., Trends Cell Biol 11:82-88,2001； Section 5.5.2of Proteins by Creighton(ISBN0-7167-2317-4)；Yu AdV Drug Deliv Rev 54:1113-1129,2002；Muller et al., Methods Enzymol 328:261-282,2000；Beck& Brodsky J Struct Biol 122:17-29,1998；Lupas Trends Biochem Sci 21:375-382, 1996；Adamson et al., Curr Opin Biotechnol 4:428-347,1993).The simplicity of coiled-coil domain Becoming design has common selection (Muller et al., the Methods of chimeric protein of determining oligomeric state Enzymol 328:261-282,2000).

In coiled-coiled structure, alpha-helix is interacted by hydrophobic residue, and the hydrophobic residue is along each spiral shell The side of rotation forms nonpolar band, and stable electrostatic phase interaction also may be present between the side chain on the band either side With.In the abcdefg heptapeptide of alpha-helix repeats, nonpolar band is limited by the hydrophobic side chain at residue a and d, wherein appointing What electrostatic interaction is predominantly located at residue e and g.Position a is most commonly that Leu, Ile or Ala, position d be usually Leu or Ala.Residue e and g are usually Glu or Gln, and Arg and Lys is also very prominent in position g.Charged residues are at position b, c and f Common, because these residues are contacted with solvent.However, this routine heptapeptide mode also has exception, and sometimes in heptapeptide It was found that Pro residue.These exceptions usually have functional meaning, and the stabilization removal including such as oligomerization domain is to allow to roll over again Folded and rearrangement, such as the refolding and rearrangement that occur in F protein.

Hundreds of coiled-coil domain sequences are known in the art, as long as it retains and other coiled-coil domains It carries out the ability of oligomerization and it does not destroy or significantly damages the function in other structures domain in PD-L2 polypeptide, then it is any suitable Sequence may be used as oligomerization domain of the invention.It, can be with as the alternative solution for using natural crimp helix domain Using artificial coiled-coil domain (Chao et al., J Chromatog B Biomed Sci Appl 715:307-329, 1998；Arndt et al., Structure 10:1235-1248,2002).Due to the height repetitive structure of coiled-coil domain, Structural domain is particularly suitable for computer modeling, because the backbone portion of each amino acid residue can parameterize, and not having to will be residual Each skeleton part of base is handled as the distinct unit with own variable.Structural domain (b) may include leucine zipper Sequence or alanine zipper sequences (Liu&Lu J Biol Chem 277:48708-48713,2002).

Have shown that coiled coil exists in the form of dimer, tripolymer, the tetramer and pentamer.In many types Albumen in find them, including transcription factor, such as, but not limited to fos, jun, c-myc, GCN4, virus fusion peptide, SNARE Compound and certain tRNA synthase etc..The coiled coil grown is found very much in albumen, such as tropomyosin, intermediate filament and spindle pole Body ingredient.Other examples are thrombospondin and cartilage oligo-substrate protein (COMP), wherein (blood platelet is anti-for connection three 2) or five (3,4 and COMP of thrombospondin) chains albumen 1 and is answered.These molecules have bouquet shape appearance, oligomerization The reason of structure may be the multivalence interaction of C- terminal domains and cell receptor.Yeast transcriptional activity factor GCN4 is super One of the eukaryotic protein for crossing 30 kinds of identifications, containing base region leucine zipper (bZIP) DNA binding motif, Ellenberger et al. (Cell 71:1223-1237,1982).BZIP dimer is a pair of continuous α spiral, they are at it Parallel coiled coil is formed on 34 residues of carboxyl terminal, and is gradually dissipated to its amino terminal to cross over DNA bound site The tap drain of point.Another example is CMP (cartilage matrix protein antigen (matrilin-1), the work separated from bovine tracheal cartilage For the homotrimer (Paulsson and Heinegard Biochem J.197:367-375,1981) of 52,000 subunit of Mr, In each subunit by vWFA1 module, single EGF structural domain, vWFA2 module and across five heptapeptides coiled-coil domain group At (Kiss et al. J.Biol.Chem.264:8126-8134,1989；Hauser and Paulsson J.Biol.Chem.269: 25747-25753,1994).Another example is cartilage oligo-substrate protein (COMP).Non- collagen glycoprotein C OMP is first soft It is accredited in bone and comes out (Hedbom, et al. J.Biol.Chem.267:6132-6136,1992).The albumen is 5 of 524kDa The same pentamer of subunit, by the end N- heptad repeat region (cc) and four subsequent epidermal growth factor (EGF) spline structure domains (EF), seven calcium binding structural domains (T3) and C- distal ball structural domain (TC) composition.The structure in domain according to this structure, COMP belong to In thrombospondin family.

In specific embodiments, coiled-coil oligomerization structural domain is leucine zipper.By leucine zipper structure The dimer that domain is formed is stablized by heptapeptide repetitive sequence, and heptapeptide repetitive sequence is named as (abcdefg)_n(see for example, McLachlan and Stewart, J.Mol.Biol.98:293,1978), wherein residue a and d is usually hydrophobic residue, and wherein d is Leucine is arranged on the same face of spiral.Residue with opposite charges typically occurs in position g and e.Therefore, by In the parallel coiled coil that two coil leucine zippers structural domains are formed, " protrusion " that is formed by the hydrophobic side chain of the first spiral It is filled out into " hole " formed between the second spiral side chain.

Illustrative leucine zipper as this paper oligomerization domain, can be derived from appointing in two seed nucleus transforming proteins Meaning, fos and jun show the product of leucine zipper motif or mouse proto-oncogene, c-myc.Leucine zipper structure Domain is necessary to bioactivity in these albumen (DNA combination).The product fos and jun of core proto-oncogene, which contain, to be preferentially formed Leucine zipper motif (O'Shea et al. the Science, 245:646,1989 of heterodimer；Turner and Tijian Science, 243:1689,1989).For example, have been displayed the leucine zipper motif of human transcription factor c-jun and c-fos with The stoichiometry of 1:1 forms stable heterodimer (see, for example, Busch and Sassone-Corsi, Trends Genetics, 6:36-40,1990；Gentz et al., Science, 243:1695-1699,1989).Although having been displayed to be formed Jun-jun homodimer, but they about 1000 times lower than jun-fos heterodimer stability.

In general, the leucine zipper motif of c-jun or c-fos is merged in the end C- of PD-L2 polypeptide.C-jun and c- The exemplary amino acid sequence of fos leucine zipper includes being respectively as follows:

In addition, the connection of PD-L2 polypeptide and leucine zipper can be directly or can be used flexible joint structural domain, Such as IgG hinge area or other p1 amino acids peptide linker (such as various length and combined glycine, serine, Soviet Union Propylhomoserin or alanine, as described in more detail below).In some cases, can by with coding proteolytic cleavage site Sequence (such as thrombin cleavage site) is merged, Lai Shixian leucine zipper and coding peptide C-end separation.

Another exemplary leucine zipper motif as oligomerization domain is derived from nucleoprotein, plays participation and makes In brewer yeast (Saccharomyces cerevisiae) transcriptional activation of the gene family of conventional nitrogen control (GCN4) metabolism because The effect of son.The albumen can dimerization, and combine containing GCN4 identification sequence promoter sequence, thus nitrogen exhaust when swash Transcription living.This structural domain is (O'Shea et al., Science 243,534-542,1989 known in the art；Harbury etc. People, Science 262,1401-1407,1993).It is capable of forming the exemplary sequence of the GCN4 leucine zipper of dimerization compound Column are appropriately selected from:

It has been found that representing the amino acid substitution of a and d residue in the synthetic peptide of GCN4 leucine zipper motif (that is, SEQ Amino acid substitution in sequence shown in ID NO:16) change the oligomerization property of leucine zipper motif.For example, working as position When all residues on a all become isoleucine, leucine zipper still forms parallel dimer.In addition to this variation Outside, when all leucine residues on the d of position also become isoleucine, it is parallel that gained peptide spontaneously forms tripolymer in the solution Coiled coil.The exemplary sequence for being capable of forming this GNC4 leucine zipper motif of tripolymer is selected from:

With all amino acid of isoleucine the position of substitution d, and with all amino acid of leucine the position of substitution a, obtain The peptide of tetramerization.The representative series for being capable of forming the leucine zipper motif of the GCN4 of the tetramer are appropriately selected from:

It is still referred to as leucine zipper motif containing these peptides replaced, as it is assumed that mechanism and biography that oligomer is formed Unite leucine zipper motif mechanism it is identical, such as GCN4 shown in above-mentioned and SEQ ID NO:16.

Optional coiled-coil domain is taken to form those of bacterial transmembrane protein of tripolymer.Suitable cross-film egg White chessman collection is adhesin (i.e. mediate adhesion to other cells or be adhered to the cell surface protein on surface), is especially that non-umbrella is viscous Attached element (for example, in oligomerization coiled coil adhesin or " Oca " family).It include with reference to text for particular sequence of the invention Those disclosed in 24 are offered, yersinia enterocolitica (Yersinia enterocolitica) adhesin is come from YadA, Neisseria meningitidis (Neisseria meningitidis) adhesin NadA, moraxelle catarrhalis (Moraxella Catarrhalis) surface protein UspA2 and other adhesins, such as from haemophilus influenzae Egypt bion The HadA adhesin of (Haemophilus influenzae biogroup aegyptius) etc. is (referring to WO2006/011060 SEQ ID NO 28-31 and 42-58).In addition, eukaryon Features of The Heat Shock Transcription Factor has coiled coil trimerising domain, Can be with single expression, and be therefore used in conjunction with the invention.

The another kind of oligomerization domain that can be used in conjunction with the invention is present in left-handed three spiral shell of referred to as collagen helix In rotation (the Proteins 5.5.3 of Creighton saves (ISBN 0-7167-2317-4)).These three spiralizations sequences are related to 1Gly²Xaa³The basic tripeptides repetitive sequence of Xaa, wherein²Xaa is usually Pro,³Xaa is usually 4-Hydroxyproline.Although this Motif is referred to as " collagen " spiral, but it is found in many albumen, rather than just collagen.Therefore, oligomerization Changing structural domain can be comprising multiple repetitive sequence motifs¹Gly²Xaa³The sequence of Xaa, the motif are folded to form helical structure, The helical structure can helical structure oligomerization corresponding in other polypeptide chains.

Collagen also provides another kind of oligomerization domain.Zhang&Chen (J Biol Chem 274:22409- 22413,1999) motif found in the non-collagen structural domain 1 (NC1) of X-type collagen is described, the motif is available In formation without the tripolymer and more advanced oligomer of three spirals.The combination of this trimerizing is high temperature stable, is not had Intermolecular disulfide bond.Therefore, oligomerization domain may include NC1 sequence.

T4 bacteriophage minority fibers albumen (fibritin) trimerising domain (foldon) (Tao et al., Structure5:789-798,1997；Gu the et al. J.Mol.Biol.337,905-915,2004), especially foldon's 27 to 30 residues in the end C- or derivatives thereof can also be used for the oligomerization of PD-L2 polypeptide.The trimerising domain can have Sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL [SEQ ID NO:25] or GSGYIPEAPRDGQAYVRKDGEWVLLSTFL [SEQ ID NO:26].Also contemplate the small modification of the structural domain.These modifications can be Cys and replace Asp 9, it is therefore an objective in phase Disulphide bridges are formed between adjacent structural domain.Other modifications of this structural domain surface amino groups acid may include the substitution of residue, with excellent Change the interaction between adjacent oligomerization domain on interface, such as hydrophobic, hydrophilic or ionic interaction or covalent bond such as two Sulphur bridge.However, other modifications of this structural domain surface amino groups acid may include the substitution of amino acid (for example, being substituted by half Guang Propylhomoserin or lysine), to generate the connection site of functional group.

In other embodiments, the tetramerization knot of viscous connection albumen (tetrabrachion) coiled-coil domain of four arms Structure domain (Stetefeld et al., Nature Structural Biology 7 (9): 772-776,2000) or derivatives thereof is used for The oligomerization of PD-L2 polypeptide.The tetramerization structural domain suitably includes sequence IINETADDIVYRLTVIIDDRYESLKNLITL RADRLMIINDNVSTILASG [SEQ IDNO:27].The sequence of coiled coil is characterized by having 3,4- hydrophobic duplicate seven The heptapeptide repetitive sequence of a residue.After a small number of corners, residue is allowed to occupy next week of quasi- (quasi-) equivalent locations Phase is three corners or 11 residues.Based on 11 duplicate presence of residue, hyperthermophilic archeobacteria sea grape hyperthermophile is come from The end C- of the viscous connection albumen of four arm of superficial layer glycoprotein of (Staphylothermus marinus) forms the coiled coil of dextrorotation Structure.It forms the tetramerization alpha-helix coiled coil stem of 70nm long, is anchored on cell membrane in the end C-.

Invention also contemplates that being used for five multimerisation domains of PD-L2 polypeptide oligomerization.Such non-limiting knot Structure domain is five multimerisation domains (Malashkevich et al., Science 274:761-765,1996) or its derivative of COMP Object.This five multimerisation domain may include sequence

LAPQMLRELQETNAALQDVRELLRQQVKQITFLKNTVMECDCG [SEQ ID NO:28].Also contemplate this The shorter construct of sequence, such as lack the CDACG motif of the end C-, wherein cysteine is in the end five multimerisation domains C- Form intermolecular disulfide bridges.

Alternatively, five multimerisation domains be tryptophan zipper (Liu J et al., Proc Natl Acad Sci USA 101: 16156-16161,2004) or derivatives thereof.Such non-limiting five multimerisation domain includes sequence SSNAKWDQWS SDWQTWNAKWDQWSNDWNAWRSDWQAWKDDWARWNQRWDNWAT[SEQ ID NO29]。

Another useful oligomerization domain is the end C- of IgA heavy chain immunoglobulin, also referred to as the tail portion α (α tp), It forms six aggressiveness.Representative six dimerizations α tp sequence length is 18 amino acid, derives from people IgA molecule.Implement at one In scheme, the tail portion α is PTHVNVSVVMAEVDGTCY [SEQ ID NO:30].However, if it is desired to can be by changing residue 1 or 2 amino acid at 5-7 (NVS) carrys out modified peptides to remove glycosylation site.For example, the asparagine (N) in position 5 can To become glutamine (Q).Alternatively, the serine (S) of position 7 can become alanine (A).Furthermore it is also possible to include IgA permanent Determine a few amino acids residue in area, such as about 4 amino acid of IgA constant region.Suitable IgA molecule packet with the useful tail portion α Include but be not limited to people IgA1, people IgA2, rabbit IgA and mouse IgA.The peptide is directly or indirectly connected to the constant knot of immunoglobulin Structure domain, for example including C_H2And C_H3The segment of structural domain.

2.3.3 the protein-protein interaction between subunit

Substitution oligomerization domain for PD-L2 polypeptide oligomerization is wherein by albumen-between different subunit polypeptides Protein-interacting and those of promote oligomerization.This oligomerization domain, such as derived from kinases A ankyrin (AKAP) The mechanism of cAMP deopendent protein kinase (PKA) and its anchoring domain (AD).Therefore, different oligomerization PD-L2 polypeptide can lead to It crosses and PD-L2 polypeptide is merged with the R subunit sequence (direct or indirect) of PKA and is generated, illustrated examples are: SHIQIPPGLTE LLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA [SEQ ID NO:31].Due to the spontaneous shape for the dimer that R subunit influences At this generates homodimer molecule.Then, another to generate by merging another PD-L2 polypeptide with the AD sequence of AKAP Kind PD-L2 peptide fusion, it includes such as sequences: QIEYLAKQIVDNAIQQ [SEQ ID NO:32].In host cell altogether When expressing the coded sequence of each in these chimeric polyeptides, dimerization R subunit provides the docking site in conjunction with AD sequence, produces Raw different oligomerization chemoattractant molecule.The binding events can further be stablized by covalent bond, such as disulfide bond.In some instances, soft Property linker residue can merge between PD-L2 polypeptide and oligomerization domain.In another example, PD-L2 polypeptide melts Conjunction can be the R subunit containing cysteine residues, and the cysteine residues being incorporated to are adjacent to the amino terminal of R subunit to promote It is covalently attached.The R subunit of this modified PKA may include such as sequence: CSHIQIPPGLTELLQGYTVEVLRQQPPDLV EFAVEYFTRLREARA [SEQ ID NO:33].Similarly, the fusion of PD-L2 polypeptide can be AD subunit, also include to AD The cysteine residues that amino terminal and carboxyl terminal are incorporated to.The representative series of the AD subunit of this modification include sequence: CGQIEYLAKQIVDNAIQQAGC [SEQ ID NO:34].

Can be used for PD-L2 polypeptide oligomerization, promote generate and be expressed as respectively PD-L2 peptide fusion two or more Other oligomerization domains of the protein-protein interaction of kind polypeptide, including such as barnase-bacillus RNA Enzyme (barnase-barnase) module (see for example, Deyev et al., Nat.Biotechnol.21:1486-1492,2003)； The use of specific protein structural domain is (see, for example, Terskikh et al., Proc Natl Acad Sci USA 94:1663- 1668,1997；And Muller et al., FEBS Lett.422:259-264,1998)；The use of specific peptide motif is (referring to example Such as, de Kruif et al., J.Biol.Chem.271:7630-7634,1996；And Muller et al., FEBS Lett.432: 45-49,1998)；And use disulphide bridges enhancing stability (de Kruif et al., J.Biol.Chem.271:7630-7634, 1996；And Schmiedl et al., Protein Eng.13:725-734,2000), and specific binding pair, such as biotin- Avidin, biotin-Streptavidin, Ag-Ab, haptens-antihapten, ligand-receptor and receptor-altogether by Body.

2.4Connector

Chimeric polyeptides of the invention may include separating or making two oligomerization knots for PD-L2 polypeptide and oligomerization domain The connector that structure domain separates.Connector generally includes any amino acid residue that cannot be expressly specified as heptapeptide repetitive sequence.Connector warp It is usually used in protein engineering field to interconnect different functional units, for example, generating derived from antibody variable light chain (VL) and variable Single chain variable fragment (scFv) construct of heavy chain (VH).They are suitble to usually in the solution with the flexibility in conformation In and be mainly made of polar amino acid residues type.Typical case (common) amino acid in flexible joint is serine and sweet ammonia Acid.Less preferably, flexible joint may also include alanine, threonine and proline.Therefore, the centre of chimeric polyeptides (intervening) connector is preferably flexible in conformation, to ensure PD-L2 polypeptide and oligomerization domain or two oligomerizations Change (without hindrance) combination of freedom of structural domain.Suitable linkers for polypeptide contemplated herein carry out those skilled in the art Say be clearly, as long as and connector be flexible in structure, it is usually other to can be in this field for connecting amino acid Any connector of sequence, i.e., they do not influence the bioactivity of PD-L2 polypeptide or the oligomerization features of oligomerization domain.

Those skilled in the art will optionally determine best connector after the routine experiment for carrying out limited quantity.It is suitable When, transition joint is such amino acid sequence, is usually made of at least one amino acid residue, usually by least two ammonia Base acid residue composition, for convenience, selects the non-critical upper limit for about 100 amino acid residues.In specific embodiments, Connector is residual by about 1 to about 50 amino acid residue or about 50 to about 100 amino acid residues, normally about 1 to about 40 amino acid Base, normally about 1 to about 30 amino acid residue composition.Particularly, in a not limiting embodiment, joint sequence is at least 50% amino acid residue is selected from proline, glycine and serine.In further non-limiting embodiments, joint sequence At least 60%, for example, at least 70%, such as 80%, more particularly 90% amino acid residue be selected from proline, glycine and Serine.In other specific embodiments, joint sequence is substantially made of polar amino acid residues；It is specific real at these It applies in scheme, at least the 50% of joint sequence, for example, at least 60%, such as 70% or 80%, more particularly 90% or at most 100% amino acid residue is selected from glycine, serine, threonine, alanine, proline, histidine, asparagine, asparagus fern Propylhomoserin, glutamine, glutamic acid, lysine and arginine.In some embodiments, joint sequence includes GG, [GGSG]_nGG、[GGGGS]_n、[SSSSG]_n、[SSSSG]_n、[AAPA]_n、[GGGKGGGG]_n、[GGGNGGGG]_n、[GGGCGGGG]_n, wherein n It is 1 to 10 integer, suitably 1 to 5, more suitably 1 to 3.

In specific embodiments, exemplary adapter can be selected from: GGGGG [SEQ ID NO:35], GGGGS [SEQ ID NO:36], SSSSG [SEQ ID NO:37], GKSSGSGSESKS [SEQ ID NO:38], GSTSGSGKSSSEGSGSTKG [SEQ ID NO:39], GSTSGSGKPGSGEGSTKG [SEQ ID NO:40], EGKSSGSGSESKEF [SEQ ID NO:41], GGSTSGSGKSSEGKG [SEQ ID NO:42], AAPA [SEQ ID NO:43].

2.5Other parts

Chimeric polyeptides of the invention may further include purification part to promote PD-L2 polypeptide-oligomerization domain embedding Fit purifying.Purification part generally comprises one section of amino acid, and recycling chimeric polyeptides can be combined by affinity.It is many pure Change part or " label " be it is known in the art, illustrated examples include biotin carboxyl carrier protein label (BCCP- mark Label), Myc- label (c-myc label), calmodulin-label, FLAG- label, HA- label, His- label (six histidines-mark Label, His6,6H), maltose-binding protein-label (MBP- label), Nus- label, cellulose binding domain-label (CBD- Label), T7 peptide-label (T7- label), ubiquitin-label, chitin-binding protein-label (CBP- label), glutathione-S- Transferase-label (GST- label), green fluorescent protein-label (GFP- label), polyglutamic acid-label, amyloid beta- Label, thioredoxin-label, S- label, Softag 1, Softag 3, staphylococcal protein A-label (albumin A-label), Streptococcus protein G-label (Protein G-label), Streptavidin-binding peptide-label (SBP- label), biotin-label, chain Mould Avidin-label and V5 label.

In some embodiments, PD-L2 polypeptide and/or oligomerization domain include immune silencing or inhibition part, institute It states immune silencing or part is inhibited to inhibit subject that any one or more of these ingredients are caused or generated with immune response. Immune silencing moiety, which can be, is glycosylated enzyme (especially glycosyl transferase) institute's specific recognition and glycosylated glycosylation position Point.Glycosylation can be N- connection or O- connection.N- connection refers to the side of carbohydrate portions and asparagine residue Chain link.Tripeptide sequence N-X-S and N-X-T (wherein X is any amino acid in addition to P) are carbohydrate portions and asparagus fern acyl Amine side chain carries out the identification sequence of enzymatic connection, these sequences are commonly referred to as " glycosylation site ".The glycosylation of O- connection refers to The connection of one of sugared N- acetylgalactosamine, galactolipin or xylose with hydroxy-amino-acid, the most common hydroxy-amino-acid are However 5-OxoPro or 5- oxylysine also can be used in serine or threonine.

3. representative chimeric polyeptides construct

Suitably, exemplary chimeric polypeptide of the invention is shown in formula (VI):

PD-L2-L-OMD_X (VI)

Wherein:

PD-L2 selected from SEQ ID NO:1 to any of 9 or with sequence shown in SEQ ID NO:1 to any of 9 Arrange and have at least 70% (and at least 71% to 99% and between all integer percents) sequence similarity or identity Polypeptide；

- L- represents key (for example, peptide bond) or peptide linker, is selected from: [GGSG]_nGG、[GGGGS]_n、[SSSSG]_n、 [SSSSG]_n、[AAPA]_n、[GGGKGGGG]_n、[GGGNGGGG]_n、[GGGCGGGG]_n, wherein n is integer of 1 to 10, suitably 1 To 5, more suitably 1 to 3；GG, GGGGG [SEQ ID NO:35], GGGGS [SEQ ID NO:36], SSSSG [SEQ ID NO: 37], GKSSGSGSESKS [SEQ ID NO:38], GSTSGSGKSSSEGSGSTKG [SEQ ID NO:39], GSTSGSGKPGSGEGSTKG [SEQ ID NO:40], EGKSSGSGSESKEF [SEQ ID NO:41], GGSTSGSGKSSEGKG [SEQ ID NO:42] and AAPA [SEQ ID NO:43]；And

OMD_XIt is selected from:

(a) trimerising domain suitably includes amino acid sequence, or be made of amino acid sequence, wherein the ammonia Base acid sequence is selected from: any one of SEQ ID NO:21,22,25 and 26, or in SEQ ID NO:21,22,25 and 26 Either one or two of shown sequence have at least 70% (and at least 71% to 99% and between all integer percents) sequence The polypeptide of similitude or identity；

(b) tetramerization structural domain suitably includes amino acid sequence, or be made of amino acid sequence, wherein the ammonia Base acid sequence is selected from: any one of SEQ ID NO:23,24 and 27, or with any in SEQ ID NO:23,24 and 27 It is a shown in sequence have at least 70% (and at least 71% to 99% and between all integer percents) sequence similarity Or the polypeptide of identity；

(c) five multimerisation domain suitably includes amino acid sequence, or be made of amino acid sequence, wherein the ammonia Base acid sequence is selected from: SEQ ID NO:28 or 29；And

(d) six multimerisation domain suitably includes amino acid sequence shown in SEQ ID NO:30 or by SEQ ID NO: The composition of amino acid sequence shown in 30.

In other embodiments, chimeric polyeptides of the invention are suitably shown in formula (VII):

PD-L2-L-OMDY-L-OMD_Z (VII)

Wherein:

PD-L2 selected from SEQ ID NO:1 to any of 9 or with sequence shown in SEQ ID NO:1 to any of 9 Arrange and have at least 70% (and at least 71% to 99% and between all integer percents) sequence similarity or identity Polypeptide；

Key (for example, peptide bond) or peptide linker are independently represented when-L- occurs every time, is selected from: [GGSG]_nGG、 [GGGGS]_n、[SSSSG]_n、[SSSSG]_n、[AAPA]_n、[GGGKGGGG]_n、[GGGNGGGG]_n、[GGGCGGGG]_n, wherein n is 1 To 10 integer, suitably 1 to 5, more suitably 1 to 3；GG, GGGGG [SEQ ID NO:35], GGGGS [SEQ ID NO: 36], SSSSG [SEQ ID NO:37], GKSSGSGSESKS [SEQ ID NO:38], GSTSGSGKSSSEGSGSTKG [SEQ ID NO:39], GSTSGSGKPGSGEGSTKG [SEQ ID NO:40], EGKSSGSGSESKEF [SEQ ID NO:41], GGSTSGSGKSSEGKG [SEQ ID NO:42] and AAPA [SEQ ID NO:43]；

OMD_YIt is oligomerization domain, forms i subunit OMD_YOligomer (OMD_Y)_i, it is appropriately selected from:

(a) dimerization domain suitably includes amino acid sequence, or be made of amino acid sequence, wherein the ammonia Base acid sequence is selected from: any one of SEQ ID NO:10,11,12,13,14,15,16,17,18,19 and 20, or and SEQ The shown sequence in any of ID NO:10,11,12,13,14,15,16,17,18,19 and 20 has at least 70% (and extremely Few 71% to 99% and between all integer percents) sequence similarity or identity polypeptide, or include oligomerization Structural domain is made of oligomerization domain, which is suitably the member of specific binding pair selected from the following: AKAP PKA-AD sequence R subunit sequence (for example, SEQ ID NO:31 and 32 or SEQ ID NO:33 and 34), biotin-antibiosis Object fibroin, biotin-Streptavidin, Ag-Ab, haptens-antihapten, ligand-receptor and receptor-co-receptor；

(b) trimerising domain suitably includes amino acid sequence, or be made of amino acid sequence, wherein the ammonia Base acid sequence is selected from: any one of SEQ ID NO:21,22,25 and 26, or in SEQ ID NO:21,22,25 and 26 Either one or two of shown sequence have at least 70% (and at least 71% to 99% and between all integer percents) sequence The polypeptide of similitude or identity；

(c) tetramerization structural domain suitably includes amino acid sequence, or be made of amino acid sequence, wherein the ammonia Base acid sequence is selected from: any one of SEQ ID NO:23,24 and 27, or with any in SEQ ID NO:23,24 and 27 It is a shown in sequence have at least 70% (and at least 71% to 99% and between all integer percents) sequence similarity Or the polypeptide of identity；

(d) five multimerisation domain suitably includes amino acid sequence, or be made of amino acid sequence, wherein the ammonia Base acid sequence is selected from: SEQ ID NO:28 or 29；And

(e) six multimerisation domain suitably includes amino acid sequence shown in SEQ ID NO:30 or by SEQ ID NO: The composition of amino acid sequence shown in 30；And

OMD_ZIt is a kind of oligomerization domain, forms j subunit OMD_ZOligomer (OMD_Z)_j, wherein j is greater than i's Integer, suitably i+1, i+2, i+3, i+4, i+5 or i+6, wherein OMD_ZIt is appropriately selected from:

(a) trimerising domain suitably includes amino acid sequence, or be made of amino acid sequence, wherein the ammonia Base acid sequence is selected from: any one of SEQ ID NO:21,22,25 and 26, or in SEQ ID NO:21,22,25 and 26 Either one or two of shown sequence have at least 70% (and at least 71% to 99% and between all integer percents) sequence The polypeptide of similitude or identity；

(b) tetramerization structural domain suitably includes amino acid sequence, or be made of amino acid sequence, wherein the ammonia Base acid sequence is selected from: any one of SEQ ID NO:23,24 and 27, or with any in SEQ ID NO:23,24 and 27 It is a shown in sequence have at least 70% (and at least 71% to 99% and between all integer percents) sequence similarity Or the polypeptide of identity；

(c) five multimerisation domain suitably includes amino acid sequence, or be made of amino acid sequence, wherein the ammonia Base acid sequence is selected from: SEQ ID NO:28 or 29；And

(d) six multimerisation domain suitably includes amino acid sequence shown in SEQ ID NO:30 or by SEQ ID NO: The composition of amino acid sequence shown in 30；And

The non-limiting example of chimeric polyeptides of the invention is as follows:

3.1 people's PD-L2 extracellular domain-L-GCN4 trimerising domains

Wherein:

- L- represents key (for example, peptide bond) or peptide linker, is selected from: [GGSG]_nGG、[GGGGS]_n、[SSSSG]_n、 [SSSSG]_n、[AAPA]_n、[GGGKGGGG]_n、[GGGNGGGG]_n、[GGGCGGGG]_n, wherein n is integer of 1 to 10, suitably 1 To 5, more suitably 1 to 3；GG, GGGGG [SEQ ID NO:35], GGGGS [SEQ ID NO:36], SSSSG [SEQ ID NO: 37], GKSSGSGSESKS [SEQ ID NO:38], GSTSGSGKSSSEGSGSTKG [SEQ ID NO:39], GSTSGSGKPGSGEGSTKG [SEQ ID NO:40], EGKSSGSGSESKEF [SEQ ID NO:41], GGSTSGSGKSSEGKG [SEQ ID NO:42] and AAPA [SEQ ID NO:43].

GCN4 trimerising domain promotes chimeric polyeptides being self-assembled into tripolymer.

3.2 people's PD-L2 extracellular domain-L-foldon trimerising domains

Wherein:

- L- represents key (for example, peptide bond) or peptide linker, is selected from: [GGSG]_nGG、[GGGGS]_n、[SSSSG]_n、 [SSSSG]_n、[AAPA]_n、[GGGKGGGG]_n、[GGGNGGGG]_n、[GGGCGGGG]_n, wherein n is integer of 1 to 10, suitably 1 To 5, more suitably 1 to 3；GG, GGGGG [SEQ ID NO:35], GGGGS [SEQ ID NO:36], SSSSG [SEQ ID NO: 37], GKSSGSGSESKS [SEQ ID NO:38], GSTSGSGKSSSEGSGSTKG [SEQ ID NO:39], GSTSGSGKPGSGEGSTKG [SEQ ID NO:40], EGKSSGSGSESKEF [SEQ ID NO:41], GGSTSGSGKSSEGKG [SEQ ID NO:42] and AAPA [SEQ ID NO:43].

Foldon trimerising domain promotes chimeric polyeptides being self-assembled into tripolymer.

3.3 people's PD-L2 extracellular domain-L-GCN4 tetramerization structural domains

Wherein:

- L- represents key (for example, peptide bond) or peptide linker, is selected from: [GGSG]_nGG、[GGGGS]_n、[SSSSG]_n、 [SSSSG]_n、[AAPA]_n、[GGGKGGGG]_n、[GGGNGGGG]_n、[GGGCGGGG]_n, wherein n is integer of 1 to 10, suitably 1 To 5, more suitably 1 to 3；GG, GGGGG [SEQ ID NO:35], GGGGS [SEQ ID NO:36], SSSSG [SEQ ID NO: 37], GKSSGSGSESKS [SEQ ID NO:38], GSTSGSGKSSSEGSGSTKG [SEQ ID NO:39], GSTSGSGKPGSGEGSTKG [SEQ ID NO:40], EGKSSGSGSESKEF [SEQ ID NO:41], GGSTSGSGKSSEGKG [SEQ ID NO:42] and AAPA [SEQ ID NO:43].

GCN4 tetramerization structural domain promotes chimeric polyeptides to be self-assembled into the tetramer.

The tetramerization structural domain of the viscous connection albumen of 3.4 people's PD-L2 extracellular domain-L-, four arm

Wherein:

- L- represents key (for example, peptide bond) or peptide linker, is selected from: [GGSG]_nGG、[GGGGS]_n、[SSSSG]_n、 [SSSSG]_n、[AAPA]_n、[GGGKGGGG]_n、[GGGNGGGG]_n、[GGGCGGGG]_n, wherein n is integer of 1 to 10, suitably 1 To 5, more suitably 1 to 3；GG, GGGGG [SEQ ID NO:35], GGGGS [SEQ ID NO:36], SSSSG [SEQ ID NO: 37], GKSSGSGSESKS [SEQ ID NO:38], GSTSGSGKSSSEGSGSTKG [SEQ ID NO:39], GSTSGSGKPGSGEGSTKG [SEQ ID NO:40], EGKSSGSGSESKEF [SEQ ID NO:41], GGSTSGSGKSSEGKG [SEQ ID NO:42] and AAPA [SEQ ID NO:43].

The tetramerization structural domain of the viscous connection albumen of four arms promotes chimeric polyeptides to be self-assembled into the tetramer.

3.5 people's PD-L2 extracellular domain-L-COMP, five multimerisation domain

Wherein:

- L- represents key (for example, peptide bond) or peptide linker, is selected from: [GGSG]_nGG、[GGGGS]_n、[SSSSG]_n、 [SSSSG]_n、[AAPA]_n、[GGGKGGGG]_n、[GGGNGGGG]_n、[GGGCGGGG]_n, wherein n is integer of 1 to 10, suitably 1 To 5, more suitably 1 to 3；GG, GGGGG [SEQ ID NO:35], GGGGS [SEQ ID NO:36], SSSSG [SEQ ID NO: 37], GKSSGSGSESKS [SEQ ID NO:38], GSTSGSGKSSSEGSGSTKG [SEQ ID NO:39], GSTSGSGKPGSGEGSTKG [SEQ ID NO:40], EGKSSGSGSESKEF [SEQ ID NO:41], GGSTSGSGKSSEGKG [SEQ ID NO:42] and AAPA [SEQ ID NO:43].

Five multimerisation domain of COMP promotes chimeric polyeptides to be self-assembled into pentamer.

3.6 people PD-L2 extracellular domains-five multimerisation domain of L-Trp zipper

Wherein:

- L- represents key (for example, peptide bond) or peptide linker, is selected from: [GGSG]_nGG、[GGGGS]_n、[SSSSG]_n、 [SSSSG]_n、[AAPA]_n、[GGGKGGGG]_n、[GGGNGGGG]_n、[GGGCGGGG]_n, wherein n is integer of 1 to 10, suitably 1 To 5, more suitably 1 to 3；GG, GGGGG [SEQ ID NO:35], GGGGS [SEQ ID NO:36], SSSSG [SEQ ID NO: 37], GKSSGSGSESKS [SEQ ID NO:38], GSTSGSGKSSSEGSGSTKG [SEQ ID NO:39], GSTSGSGKPGSGEGSTKG [SEQ ID NO:40], EGKSSGSGSESKEF [SEQ ID NO:41], GGSTSGSGKSSEGKG [SEQ ID NO:42] and AAPA [SEQ ID NO:43].

Five multimerisation domain of tryptophan zipper promotes chimeric polyeptides to be self-assembled into pentamer.

3.7 people PD-L2 extracellular domain-L- α tp, six multimerisation domain

Wherein:

- L- represents key (for example, peptide bond) or peptide linker, is selected from: [GGSG]_nGG、[GGGGS]_n、[SSSSG]_n、 [SSSSG]_n、[AAPA]_n、[GGGKGGGG]_n、[GGGNGGGG]_n、[GGGCGGGG]_n, wherein n is integer of 1 to 10, suitably 1 To 5, more suitably 1 to 3；GG, GGGGG [SEQ ID NO:35], GGGGS [SEQ ID NO:36], SSSSG [SEQ ID NO: 37], GKSSGSGSESKS [SEQ ID NO:38], GSTSGSGKSSSEGSGSTKG [SEQ ID NO:39], GSTSGSGKPGSGEGSTKG [SEQ ID NO:40], EGKSSGSGSESKEF [SEQ ID NO:41], GGSTSGSGKSSEGKG [SEQ ID NO:42] and AAPA [SEQ ID NO:43].

Six multimerisation domain of α tp promotes chimeric polyeptides to be self-assembled into six aggressiveness.

3.8 people's PD-L2 extracellular domain-L-Fc dimerization domain-L-foldon trimerising domains

Wherein:

Key (for example, peptide bond) or peptide linker are independently represented when-L- occurs every time, is selected from: [GGSG]_nGG、 [GGGGS]_n、[SSSSG]_n、[SSSSG]_n、[AAPA]_n、[GGGKGGGG]_n、[GGGNGGGG]_n、[GGGCGGGG]_n, wherein n is 1 To 10 integer, suitably 1 to 5, more suitably 1 to 3；GG, GGGGG [SEQ ID NO:35], GGGGS [SEQ ID NO: 36], SSSSG [SEQ ID NO:37], GKSSGSGSESKS [SEQ ID NO:38], GSTSGSGKSSSEGSGSTKG [SEQ ID NO:39], GSTSGSGKPGSGEGSTKG [SEQ ID NO:40], EGKSSGSGSESKEF [SEQ ID NO:41], GGSTSGSGKSSEGKG [SEQ ID NO:42] and AAPA [SEQ ID NO:43].

The combination of Fc dimerization domain and foldon trimerising domain promotes chimeric polyeptides to be self-assembled into six aggressiveness.

3.9 people's PD-L2 extracellular domain-L-Fc dimerization domain-L-GCN4 tetramerization structural domains

Wherein:

Key (for example, peptide bond) or peptide linker are independently represented when-L- occurs every time, is selected from: [GGSG]_nGG、 [GGGGS]_n、[SSSSG]_n、[SSSSG]_n、[AAPA]_n、[GGGKGGGG]_n、[GGGNGGGG]_n、[GGGCGGGG]_n, wherein n is 1 To 10 integer, suitably 1 to 5, more suitably 1 to 3；GG, GGGGG [SEQ ID NO:35], GGGGS [SEQ ID NO: 36], SSSSG [SEQ ID NO:37], GKSSGSGSESKS [SEQ ID NO:38], GSTSGSGKSSSEGSGSTKG [SEQ ID NO:39], GSTSGSGKPGSGEGSTKG [SEQ ID NO:40], EGKSSGSGSESKEF [SEQ ID NO:41], GGSTSGSGKSSEGKG [SEQ ID NO:42] and AAPA [SEQ ID NO:43].

The combination of Fc dimerization domain and GCN4 tetramerization structural domain promotes chimeric polyeptides to be self-assembled into eight aggressiveness.

3.10 people's PD-L2 extracellular domain-L-Fc dimerization domain-L-COMP, five multimerisation domain

Wherein:

Key (for example, peptide bond) or peptide linker are independently represented when-L- occurs every time, is selected from: [GGSG]_nGG、 [GGGGS]_n、[SSSSG]_n、[SSSSG]_n、[AAPA]_n、[GGGKGGGG]_n、[GGGNGGGG]_n、[GGGCGGGG]_n, wherein n is 1 To 10 integer, suitably 1 to 5, more suitably 1 to 3；GG, GGGGG [SEQ ID NO:35], GGGGS [SEQ ID NO: 36], SSSSG [SEQ ID NO:37], GKSSGSGSESKS [SEQ ID NO:38], GSTSGSGKSSSEGSGSTKG [SEQ ID NO:39], GSTSGSGKPGSGEGSTKG [SEQ ID NO:40], EGKSSGSGSESKEF [SEQ ID NO:41], GGSTSGSGKSSEGKG [SEQ ID NO:42] and AAPA [SEQ ID NO:43].

The combination of five multimerisation domain of Fc dimerization domain and COMP promotes chimeric polyeptides to be self-assembled into ten aggressiveness.

3.11 people PD-L2 extracellular domain-L-Fc dimerization domain-L- α tp, six multimerisation domain

Wherein:

Key (for example, peptide bond) or peptide linker are independently represented when-L- occurs every time, is selected from: [GGSG]_nGG、 [GGGGS]_n、[SSSSG]_n、[SSSSG]_n、[AAPA]_n、[GGGKGGGG]_n、[GGGNGGGG]_n、[GGGCGGGG]_n, wherein n is 1 To 10 integer, suitably 1 to 5, more suitably 1 to 3；GG, GGGGG [SEQ ID NO:35], GGGGS [SEQ ID NO: 36], SSSSG [SEQ ID NO:37], GKSSGSGSESKS [SEQ ID NO:38], GSTSGSGKSSSEGSGSTKG [SEQ ID NO:39], GSTSGSGKPGSGEGSTKG [SEQ ID NO:40], EGKSSGSGSESKEF [SEQ ID NO:41], GGSTSGSGKSSEGKG [SEQ ID NO:42] and AAPA [SEQ ID NO:43].

The combination of six multimerisation domain of Fc dimerization domain and α tp promotes chimeric polyeptides to be self-assembled into ten dimers.

4. the generation of chimeric polyeptides

PD-L2 polypeptide-oligomerization domain chimera of the invention can be prepared by chemical synthesis or recombination method.It is logical It often, can be by expressing the recombination of encoding chimera polypeptide in a suitable host cell although any suitable method can be used Construct prepares chimeric polyeptides.Suitable host cell includes, for example, insect cell is (for example, Aedes aegypti (Aedes Aegypti), clover clover (Autographa californica), silkworm (Bombyx mori), Drosophila melanogaster (Drosophila melanogaster), fall army worm (Spodoptera frugiperda) and cabbage looper (Trichoplusia ni), mammalian cell are (for example, people, non-human primate, horse, ox, sheep, dog, cat and grinding tooth Animal (for example, hamster), avian cell (for example, chicken, duck and goose), bacterium (for example, Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis) and streptococcus (Streptococcus spp.)), yeast cells (for example, Saccharomyces cerevisiae (Saccharomyces cerevisiae), Candida albicans (Candida albicans), malt Candida (Candida maltosa), Hansenula polymorpha (Hansenula polymorphs), fragile kluyveromyces (Kluyveromyces fragilis), Kluyveromyces lactis (Kluyveromyces lactis), Pichia pastoris (Pichia Guillerimondii), pichia pastoris yeast (Pichia pastoris), schizosaccharomyces pombe (Schizosaccharomyces pombe) and Yarrowia lipolytica (Yarrowia lipolytica)), tetrahymena cell (example Such as, tetrahymena thermophila (Tetrahymena thermophile)) or combinations thereof.Many suitable insect cells and mammal Cell is well known in the art.Suitable insect cell includes, such as Sf9 cell, Sf21 cell, Tn5 cell, applies The S2 of resistance to moral cell and High Five cell (the clone and separate object from parent's cabbage looper BTI-TN-5B1-4 cell line (Invitrogen)).Suitable mammalian cell includes, such as Chinese hamster ovary (CHO) cell, human embryonic kidney cells (HEK293 cell, usually conversion have the Adenovirus Type 5 DNA of shearing), NIH-3T3 cell, 293-T cell, Vero cell, HeLa Cell, PERC.6 cell (ECACC deposit number 96022940), Hep G2 cell, MRC-5 (ATCC CCL-171), WI-38 (ATCC CCL-75), fetus rhesus macaque pneumonocyte (ATCC CL-160), Madin-Darby ox kidney (" MDBK ") cell, Madin-Darby dog kidney (" MDCK ") cell is (for example, MDCK (NBL2), ATCC CCL34；Or MDCK 33016, DSM ACC 2219), baby hamster kidney (BHK) cell, such as BHK21-F, HKCC cell.Suitable avian cells include, such as chicken embryo tire is dry Cell (for example,Cell), chicken embryo fibroblasts, chicken embryonic genital cell, duck cell (such as AGE1.CR and AGE1.CR.pIX cell line (ProBioGen), they are in such as Vaccine 27:4975-4982 (2009) and WO2005/ Described in 042728), EB66 cell etc..

Suitable insect cell expression system (such as rhabdovirus system) is known to the skilled in the art, and is described In such as Summers and Smith, Texas Agricultural Experiment Station Bulletin No.1555 (1987) in.It can be in a kit form from especially for baculoviral/insertion cell expression system material and method Invitrogen, San Diego Calif are commercially available.Avian cells expression system is also known to the skilled in the art, And it is described in such as U.S. Patent number 5,340,740；5,656,479；5,830,510；6,114,168；With 6,500,668； European patent number EP 0787180B；European Patent Application No. EP03291813.8；WO 03/043415；With WO 03/076601. Similarly, bacterium and mammalian cell expression system are also known in the art, and are described in such as Yeast Genetic Engineering (Barr et al. chief editor 1989) London Butterworth.

Conventional method can be used and prepare the recombinant precursor for encoding chimeric polyeptides of the present invention in suitable carrier.For It is known in this field and conventional that some suitable carriers of recombinant protein are expressed in insect or mammalian cell.It is suitable to carry Body can contain various ingredients, including but not limited to one or more of: replication orgin；Selectable marker gene；It is a kind of Or a variety of expression control elements, as transcriptional control element (for example, promoter, enhancer, terminator) and/or one or more turn over Translate signal；With in selected host cell for target secretory pathway signal sequence or leader (for example, mammal come Source comes from heterologous mammal or non-mammalian species).For example, in order to be expressed in insect cell, suitable rod-shaped disease Malicious expression vector, as pFastBac (Invitrogen) can be used for generating recombinant baculovirus particle.Expand baculovirus particles And for infected insect cell to express recombinant protein.In order to express in mammalian cells, use will drive construct to exist The carrier (for example, Chinese hamster ovary cell) expressed in required mammalian host cell.

Any suitable method purifying chimeric polyeptides can be used.Appropriate method for purifying required albumen is this field It is well known, including precipitating and various types of chromatographies, such as hydrophobic interaction, ion exchange, affine, chelating and size row Resistance.Two or more in these or other appropriate method can be used to generate suitable purification schemes.If desired, embedding Closing polypeptide may include the purification part or " label " for promoting purifying, as described in chapters and sections 3.5.It can be by chelate chromatography or affine Chromatography easily for example purifies the polypeptide of this tape label from conditioned media.

5. the polypeptide complex based on chimeric polyeptides

Chimeric polyeptides of the invention can under suitable conditions self assembly to form polypeptide complex.Therefore, of the invention It further include the method for generating polypeptide complex, wherein this method includes: (example under conditions of suitably forming polypeptide complex Such as, in aqueous solution chimeric polyeptides of the invention) are combined, it is compound thus to generate the polypeptide comprising three or more chimeric polyeptides Object, it is characterised in that at least one functional activity with PD-L2, for example (,) it is described herein.In general, chimeric polyeptides are water-soluble in buffering (for example, pH about 5 to about 9) self assembly in liquid.If desired, mild Denaturing can be used, for example, by introduce urea, A small amount of organic solvent or heating, are denaturalized chimeric polyeptides leniently, to promote folding and self assembly again.

Any suitable chimeric polyeptides preparation can be used in this approach.For example, containing the item of required chimeric polyeptides Part cell culture medium can be used in this method.It is preferable, however, that in this approach using the chimeric polyeptides of purifying.

6. composition

The present invention further provides compositions, including pharmaceutical composition, and it includes retouch extensively with elsewhere herein above The polypeptide complex or chimeric polyeptides stated or the nucleic acid construct that chimeric polyeptides or compound can be expressed, and optionally pharmaceutically may be used The carrier or adjuvant of receiving.Representative compositions may include buffer, according to the required purposes of chimeric polyeptides or compound into Row selection, and may also include other substances for being suitable for desired use.When desired use is to adjust immune response (including Th1 Immune response) when, this composition is referred to as " immunological regulation " or " immune regulative " composition.These compositions include prevention Composition (that is, the composition applied to prevent Th1 related disease or illness) is to therapeutic combination (that is, being that treatment Th1 is related Disease or illness and the composition applied).Therefore, immune regulation composite of the invention can be applied to recipient, to be used for Prevention, improvement, alleviation or therapeutic purposes.

Those skilled in the art can be readily selected the appropriate buffer for being suitable for desired use, and numerous kinds are at this It is well-known in field.In some cases, composition may include pharmaceutically acceptable excipient, therein a variety of to be It is known in the art, and without being discussed in detail herein.Pharmaceutically acceptable excipient fills in various publications Divide description, including such as A.Gennaro (2000) " Remington:The Science and Practice of Pharmacy " the 20th edition Lippincott, Williams, &Wilkins；Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C.Ansel et al. edits the 7th edition supplement, Lippincott, Williams, & Wilkins；And Handbook of Pharmaceutical Excipients (2000) A.H.Kibbe et al. edits third Version supplement Amer.Pharmaceutical Assoc.

Pharmaceutical composition of the invention can be suitable for injection application by way of, the preparation shape suitable for being orally ingested Formula (for example, capsule, tablet, capsule, elixir), the ointment suitable for local application, the form of frost or lotion, suitable for the shape of eye drip delivering Formula sucks the aerosol form (such as passing through nasal inhalation or oral sucking) of application or suitable for parenteral administration suitable for passing through Form (i.e. subcutaneous, intramuscular or intravenous injection).

The active constituent such as adjuvant or biological response modifier of supplement can also mix in pharmaceutical composition of the invention.Although Adjuvant may include in pharmaceutical composition of the invention, but composition is not necessarily required to comprising adjuvant.In this case, may be used To avoid due to using reactionogenicity problem caused by adjuvant.

In general, adjuvanticity includes but is not limited to that (quantitative or qualitative) increases under the background of pharmaceutical composition of the invention The ability of the immune response of immunogenic components (for example, chimeric polyeptides or compound of the invention) induction in strong composition.This The dosage or level of immunological regulation component needed for generating immune response (including Th1 immune response) can be reduced, and/or is reduced Immune time or frequency needed for immune response needed for generating.

Any suitable adjuvant may include in pharmaceutical composition of the invention.It is, for example, possible to use aluminium base adjuvants.Properly Aluminium base adjuvant include but is not limited to aluminium hydroxide, aluminum phosphate and combinations thereof.Other for the aluminium base adjuvant that can be used specifically show Example is described in european patent number 1216053 and U.S. Patent number 6,372,223.Other suitable adjuvants include that Freund is incomplete Adjuvant and Freund's complete adjuvant (Difco Laboratories, Detroit, Mich.)；Merck adjuvant 65 (Merck and Company, Inc., Rahway, NJ)；AS-2 (SmithKline Beecham, philadelphia, pa)；Aluminium salt, such as hydrogen-oxygen Change alumina gel (alum) or aluminum phosphate；The salt of calcium, iron or zinc；The insoluble suspension of acylated tyrosine；Acylated sugar；Cation or The polysaccharide of anionic derivative；Polyphosphazene；Biodegradable microballoon；Monophosphoryl lipid A and quil A；Oil in water emulsion, including Described in european patent number 0399843, U.S. Patent number 7,029,678 and PCT Publication WO2007/006939 those；With/ Or other cell factors, such as GM-CSF or interleukin 2, -7 or -12, granulocyte-macrophage colony stimutaing factor (GM-CSF), tumor necrosis factor (TNF) monophosphoryl lipid A (MPL), cholera toxin (CT) or its composition subunit, thermally labile Enterotoxin (LT) or its composition subunit, Toll-like receptor ligand adjuvant such as lipopolysaccharides (LPS) and its derivative are (for example, single phosphinylidyne Lipid A and 3D-MPL), flavivirus NS 1 and muramyl dipeptide (MDP).

Pharmaceutical composition of the invention can be provided in kit.Kit may include helping to implement the method for the present invention Other components, such as application device, buffer and/or diluent.Kit may include the container for accommodating various components With the specification for using reagent constituents in the method for the present invention.In general, kit includes using immuno-modulating composition of the invention The specification of object, immune regulation composite are used alone or are used together with diagnosticum, such as described herein.

Polypeptide complex of the invention can be used for enhancing the immune response to immunomodulator, including Disease associated antigens (such as antigen of tumour antigen and pathogenic organism) and antigen binding molecules.

The present invention considers to correspond at least the one of interest target antigen using any antigen in the present compositions Part, and be used to stimulate the immune response for being directed to target antigen.This antigen can be soluble form (such as peptide, polypeptide or Can be by expression of peptides or the nucleic acid molecules of polypeptide) or in the form of being full cell or attenuated pathogens preparation (for example, attenuated virus Or bacterium) or can be presented by antigen presenting cell, as described in more detail below.

6.1 antigen

Target antigen for use in the present invention can be any kind of biomolecule, including for example simple intermediate supersession Object, sugar, lipid and hormone and macromolecular, such as complex carbohydrate, phosphatide, nucleic acid, polypeptide and peptide.Target antigen is optional The exogenous antigen of the endogenous antigen or host's external source that are generated from host.Suitable endogenous antigen includes but is not limited to cancer Or tumour antigen.Cancer or the non-limiting example of tumour antigen include from the antigen selected from following cancer or tumour: ABL1 Proto-oncogene, AIDS associated cancer, acoustic neurinoma, acute lymphoblastic leukemia, acute myeloid leukaemia, adenoid cystic carcinoma, The soft part sarcoma of adrenocortical carcinoma, agnogenic myeloid metaplasia, alopecia, alveolar, cancer of anus, angiosarcoma, aplastic Anaemia, astrocytoma, asynergy-capillary dilation, (skin) basal-cell carcinoma, bladder cancer, osteocarcinoma, intestinal cancer, brain Dry glioma, brain and central nerve neuroma, breast cancer, central nerve neuroma, benign tumour, cervical carcinoma, children's brain are swollen It is tumor, childhood cancer, leukemia of children, children soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, slow Property myelogenous leukemia, colorectal cancer, skin T cell lymphoma, dermatofibrosarcoma-protrusion, Hypertrophic small-circle-cell Tumour, duct carcinoma, endocrine cancer, carcinoma of endometrium, ependymoma, the cancer of the esophagus, Ewing sarcoma, cholangiocarcinoma, cancer eye, Eye: melanoma, retinoblastoma, carcinoma of fallopian tube, Fanconi anemia, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, Gastrointestinal carcinoid tumors, urogenital neoplasm, germinoma, gestational trophoblastic disease, glioma, gynecologic cancer Disease, Malignancy, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, heredity breast cancer, histiocytosis, suddenly Odd gold disease, human papilloma virus, vesicular mole, hypercalcinemia, hypopharyngeal cancer, intraocular melanoma, islet-cell carcinoma, Ka Boxishi meat Tumor, kidney, langerhans cell histiocytosis, laryngocarcinoma, leiomyosarcoma, leukaemia, Li-Fu Laomeini syndrome, Lip cancer, embryonal-cell lipoma, liver cancer, lung cancer, lymphedema, lymthoma, Hodgkin lymphoma, non-Hodgkin lymphoma, cancer of male breast, Pernicious rhabdoid tumor kidney neoplasms, medullary substance pulmonary blastoma, melanoma, Merkel cell cancer, celiothelioma, metastatic carcinoma, mouth cancer, MEN,muitiple endocrine neoplasms, mycosis fungoides, myelodysplastic syndrome, myeloma, myeloproliferative disease, rhinocarcinoma, nose Pharynx cancer, the nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen breakage syndrome, nonmelanoma skin cancer, Non-small cell lung cancer (NSCLC), cancer eye, cancer of the esophagus, carcinoma of mouth, oropharyngeal cancer, osteosarcoma, fistulization oophoroma, cancer of pancreas, nasal sinus Cancer, parathyroid carcinoma, carcinoma of parotid gland, carcinoma of penis, peripheral neuroectodermal tumor, hypophysis cancer, polycythemia vera, prostate Cancer, rare cancer and related disease, clear-cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson are comprehensive Sign, salivary-gland carcinoma, sarcoma, neurinoma, Sezary syndrome, cutaneum carcinoma, Small Cell Lung Cancer (SCLC), carcinoma of small intestine, soft tissue Sarcoma, tumor of spinal cord, (skin) squamous cell carcinoma, gastric cancer, synovial sarcoma, carcinoma of testis, thymic carcinoma, thyroid cancer, (bladder) move Row cell cancer, (kidney-pelvis -/- ureter) transitional cell carcinoma, choriocarcinoma, carcinoma of urethra, urinary system cancer, urinary tract patch (uroplakins), sarcoma of uterus, uterine cancer, carcinoma of vagina, carcinoma of vulva, Waldenstrom's macroglobulinemia, wilms' tumor.Certain In embodiment, cancer or tumour are related to melanoma.The illustrated examples of melanic related antigen include melanocyte point Change antigen (for example, gp100, MART, Melan-A/MART-1, TRP-1, Tyros, TRP2, MC1R, MUC1F, MUC1R or its group Close) and melanoma specific antigen (for example, BAGE, GAGE-1, gp100In4, MAGE-1 are (for example, GenBank accession number X54156 and AA494311), MAGE-3, MAGE4, PRAME, TRP2IN2, NYNSO1a, NYNSO1b, LAGE1, p97 melanin Tumor antigen (for example, GenBank accession number M12154), p5 albumen, gp75, oncofetal antigen, GM2 and GD2 gangliosides, cdc27、p21ras、gp100^Pmel117Or combinations thereof.Other tumour specific antigens include but is not limited to: etv6, aml1, close ring Albumen b (acute lymphoblastic leukemia)；Ig- idiotype (B cell lymphoma)；CAM 120/80, α-catenin, the β-chain of rings Albumen, γ-catenin, p120ctn (glioma)；P21ras (bladder cancer)；P21ras (cholangiocarcinoma)；MUC family, HER2/neu, c-erbB-2 (breast cancer)；P53, p21ras (cervical carcinoma)；P21ras, HER2/neu, c-erbB-2, MUC family, Cripto-1 albumen, Pim-1 albumen (colon cancer)；Colorectal associated antigen (CRC)-CO17-1A/GA733, (colon is straight by APC Intestinal cancer)；Carcinomebryonic antigen (CEA) (colorectal cancer；Choriocarcinoma)；Cyclophilin b (cell carcinoma)；HER2/neu,c- ErbB-2, ga733 glycoprotein (gastric cancer)；α-fetoprotein (hepatocellular carcinoma)；Imp-1, EBNA-1 (Hodgkin lymphoma)；CEA, MAGE-3, NY-ESO-1 (lung cancer)；Cyclophilin b (the derivative leukaemia of lymphocyte)；MUC family, p21ras (myeloma)； HER2/neu, c-erbB-2 (non-small cell lung cancer)；Imp-1, EBNA-1 (nasopharyngeal carcinoma)；MUC family, HER2/neu, c-erbB- 2, MAGE-A4, NY-ESO-1 (oophoroma)；Prostate-specific antigen (PSA) and its epitope PSA-1, PSA-2 and PSA- 3, PSMA, HER2/neu, c-erbB-2, ga733 glycoprotein (prostate cancer)；HER2/neu, c-erbB-2 (kidney)；Virus produces Object such as human papilloma virus toxalbumin (cervix and esophageal squamous cell carcinoma)；NY-ESO-1 (carcinoma of testis)；And HTLV-1 epitope (T cell leukaemia).

Suitably, exogenous antigen is selected from pathogenic organism.Exemplary pathogenic organism include but is not limited to virus, bacterium, Fungi, parasitic animal and plant, algae and protozoan and amoeba.The illustrated examples of virus include being responsible for the virus of certain diseases, this A little diseases include but is not limited to: (such as GenBank is stepped on for measles,mumps,rubella, polio, hepatitis A, hepatitis B Record E02707) and hepatitis C (such as GenBank accession number E06890) and other hepatitis virus, influenza, adenovirus (such as 4 With 7 types), rabies (such as GenBank accession number M34678), yellow fever virus, Epstein-Barr virus and other herpesvirals such as papillomatosis Poison, Ai Bola virus, influenza virus, encephalitis B (such as GenBank accession number E07883), dengue fever (such as GenBank accession number M24444), hantavirus, sendai virus, Respiratory Syncytial Virus(RSV), orthomyxovirus (othromyxoviruses), vesiculovirus mouth Scorching virus, visna virus, cytomegalovirus and human immunodeficiency virus (HIV) (such as GenBank accession number U18552).Any suitable antigen derived from these viruses is used equally for implementing the present invention.For example, illustrative derived from HIV O retrovirus antigens include but is not limited to: the antigen such as gene product of gag, pol and env gene, Nef albumen, reverse transcriptase With other HIV ingredients.The illustrated examples of hepatitis virus antigen include but is not limited to: S, M and L of antigen such as hepatitis type B virus Albumen, the pro-S antigen of hepatitis type B virus and other hepatitis (such as first, second and hepatitis C) virus composition (such as hepatitis C Viral RNA).The illustrated examples of influenza antigen include but is not limited to: antigen such as hemagglutinin and neuraminidase and other Influenza virus ingredient.The illustrated examples of measles virus antigens include but is not limited to: antigen such as Fusion protein of measles virus and its His measles virus ingredient.The illustrated examples of rubella virus antigen include but is not limited to: antigen such as albumen E1 and E2 and other Rubella virus ingredient；Wheel virus antigen such as VP7sc and other rotavirus components.The illustrated examples of cytomegalovirus antigen Including but not limited to: antigen such as envelope glycoprotein B and other cytomegalovirus antigen ingredients.Respiratory syncytial viral antigens are said Bright property example includes antigen such as RSV fusion protein, M2 albumen and other respiratory syncytial viral antigens ingredients.Herpes simplex virus The illustrated examples of antigen include but is not limited to: antigen such as early protein, glycoprotein D and other herpes simplex virus antigens immediately Ingredient.The non-limiting example of varicellazoster virus antigen includes antigen such as 9PI, gpII and other varicella zosters disease Malicious antigenic component.The non-limiting example of japanese encephalitis virus antigen includes antigen such as albumen E, M-E, M-E-NS1, NS1, NS1- NS2A, 80%E and other japanese encephalitis virus antigenic components.The representative example of Rabies Virus Antigen includes but is not limited to: Antigen such as rabies glycoproteins, rabies nucleoprotein and other Rabies Virus Antigen ingredients.The explanation of papilloma virus antigens Property example includes but is not limited to: L1 and L2 capsid protein and with the Cancer-Related E6/E7 antigen of uterine neck, other viral antigens show Example refers to the Fundamental Virology second edition Fields, B.N. and Knipe, and D.M. is compiled, 1991, Raven Press, New York.

The illustrated examples of fungi include Acremonium (Acremonium spp.), aspergillus (Aspergillus Spp.), basidiobolus haptosporus (Basidiobolus spp.), Bipolaris (Bipolaris spp.), Blastomyces dermatitidis (Blastomyces dermatidis), Mycotoruloides (Candida spp.), Cattell branch spore Saksenaea vasiformis (Cladophialophora Carrionii), posadasis spheriforme (Coccidioides immitis), Conidiobolus (Conidiobolus spp.), cryptococcus Belong to (Cryptococcus spp.), Curvularia lunata category (Curvularia spp.), Epidermophyton (Epidermophyton Spp.), Exophiala jeanselmei (Exophiala jeanselmei), prominent navel helminthosporium spp (Exserohilum spp.), close Color fungi (Fonsecaea compacta), fonsecaea pedrosoi (Fonsecaea pedrosoi), Fusarium oxysporum (Fusarium oxysporum), Beancurd sheet reaping hook mattress (Fusarium solani), geotrichum candidum (Geotrichum candidum), Histoplasma capsulatum var. cap sulatum (Histoplasma capsulatum var.capsulatum), Histoplasma capsulatum Du Shi mutation (Histoplasma capsulatum var.duboisii), Hortaea werneckii, Lacazia Loboi, seat chamber double born of the same parents bacterium (Lasiodiplodia theobromae), leptosphaeria senegalensis (Leptosphaeria Senegalensis), grey Madura branch bacterium (Madurella grisea), foot Madura mycomycete (Madurella Mycetomatis), Malassezia furfur (Malassezia furfur), microspore Pseudomonas (Microsporum spp.), sieve The new tortoise-shell shaped bacterium (Neotestudina rosatii) of Sa ladder, Onychocola canadensis, Brazilian yeast (Paracoccidioides brasiliensis), Phialophora verrucosa (Phialophora verrucosa), piedraia hortai (Piedraia hortae), Piedra iahortae, tinea versicolor (Pityriasis versicolor), Pseudallesheria boydii, pyrenochaeta romeroi (Pyrenochaeta romeroi), Rhizopus arrhizus (Rhizopus Arrhizus), small broom sample mould (Scopulariopsis brevicaulis), it is double between capital spore (Scytalidium Dimidiatum), Sporothrix schenckii (Sporothrix schenckii), trichophyton (Trichophyton spp.), hair Spore Pseudomonas (Trichosporon spp.), zygomycete (Zygomcete fungi), absidia corymbifera (Absidia Corymbifera), Rhizomucor pusillus (Rhizomucor pusillus) and Rhizopus arrhizus (Rhizopus arrhizus).Cause This, the illustrative fungal antigen that can be used for the present composition and method includes but is not limited to: Candida antigenic component； Tissue milk Pseudomonas fungal antigen such as Heat Shock Protein 60 (HSP60) and other tissue milk Pseudomonas fungal antigen ingredients；Cryptococcus Fungal antigen such as capsular polysaccharide and other Cryptococcus fungal antigen ingredients；Coccidioides fungal antigen such as spherula antigen and Other Coccidioides fungal antigen ingredients；And tinea fungal antigen such as trichophytin and other Coccidioides fungal antigens at Point.

The illustrated examples of bacterium include being responsible for the bacterium of disease, including but not limited to: diphtheria (such as corynebacterium diphtheriae (Corynebacterium diphtheriae)), pertussis (such as pertussis byrd bacteria (Bordetella pertussis), GenBank accession number M35274), tetanus (such as clostridium tetani (Clostridium tetani), GenBank accession number M64353), tuberculosis (such as tubercle bacillus (Mycobacterium tuberculosis)), (such as influenza is bloodthirsty for bacterial pneumonia Bacillus (Haemophilus influenzae)), cholera (such as comma bacillus (Vibrio cholerae)), anthrax (such as anthrax bar Bacterium (Bacillus anthracis)), typhoid fever, the plague, shigellosis (such as Shigella dysenteriae (Shigella Dysenteriae)), botulismus (such as clostridium botulinum (Clostridium botulinum)), salmonellosis (such as GenBank Accession number L03833), peptic ulcer (such as helicobacter pylori (Helicobacter pylori)), legionaires' disease, Lyme disease (such as GenBank accession number U59487)；Other pathogens include Escherichia coli (Escherichia coli), C.perfringens (Clostridium perfringens), pseudomonas aeruginosa (Pseudomonas aeruginosa), staphylococcus aureus (Staphylococcus aureus) and streptococcus pyogenes (Streptococcus pyogenes).Therefore, it can be used for the present invention Composition and the bacterial antigens of method include but is not limited to: Pertussis bacteria antigen such as pertussis toxin, filamentous hemagglutinin, one hundred days Cough bacillus adhesin (pertactin), FM2, FIM3, adenyl cyclase and other Pertussis bacteria antigenic components；Diphtheria is thin Bacterium antigen such as diphtheria toxin or toxoid and other diphtheria bacterial antigens ingredients；Tetanus bacterial antigens such as tetanus toxin or class Toxin and other tetanus bacterial antigens ingredients, streptococcus bacterium antigen such as M albumen and other streptococcus bacterium antigenic components；Leather Blue negative bacillus bacterial antigens such as lipopolysaccharides and other gram-negative bacteria antigenic components；Tubercle bacillus (Mycobacterium Tuberculosis) bacterial antigens such as mycolic acid, Heat Shock Protein 65 (HSP65), 30kDa kDa major secretory protein, antigen 85A With other antigen of mycobacterium ingredients；Helicobacter pylori (Helicobacter pylori) bacterial antigens ingredient, pneumococcus are thin Bacterium antigen such as pneumolysin, pneumococcal capsular polysaccharide and other pneumococcus bacterial antigens ingredients；Influenza is bloodthirsty Bacillus (Haemophilus influenz) bacterial antigens such as capsular polysaccharide and other Hinfluenzae bacterium antigenic components；Charcoal Subcutaneous ulcer bacterial antigens such as anthrax protective antigen and other anthracia bacterium antigenic components；Richettsia bacterial antigens such as rompA and other Richettsia bacterial antigens ingredient.Bacterial antigens described herein also include any other bacterium, mycobacteria, mycoplasma, Garrick Secondary body or CHLA Casset.

Protozoic illustrated examples include being responsible for the protozoan of disease, and including but not limited to: plasmodium is (such as GenBank accession number X53832), hookworm, filaria volvulus (such as GenBank accession number M27807), (such as GenBank is stepped on blood fluke Record LOS 198), toxoplasm, trypanosome, Leishmania, giardia lamblia stiles (GenBank accession number M33641), amoeba, silk Worm (such as GenBank accession number J03266), Borellia (borreliosis) and trichina.Therefore, it can be used for combination of the present invention Object and the protozooal antigens of method include but is not limited to: plasmodium falciparum (plasmodium falciparum) antigen is such as split Grow sub- surface antigen, sporozoite surface antigen, ring sporozoite antigen, gametocyte/subsurface antigen, blood stage antigens Pf155/RESA and other plasmodium antigens ingredients；Tox Antigen such as SAG-1, p30 and other Tox Antigen ingredients；Blood is inhaled Worm antigen such as glutathione-S-transferase, paramyosinogen and other schistosome antigen ingredients；Leishmania major and other Leishmania antigen such as gp63, lipophosphoglycan and its GAP-associated protein GAP and other leishmania antigen ingredients；And kirschner Trypanosome (trypanosoma cruzi) antigen such as 75-77kDa antigen, 56kDa antigen and other Trypanosoma antigens ingredients.

Present invention also contemplates that toxic components are used as antigen.The illustrated examples of toxin include but is not limited to staphylococcus intestines Toxin, toxic shock syndrome toxin；O retrovirus antigens (antigen as being derived from HIV), streptococcal antigens, grape ball Bacterium enterotoxin-A (SEA), staphylococcal enterotoxin-B (SEB), staphylococcal enterotoxin_1-3(SE_1-3), staphylococcal enterotoxin-D (SED), staphylococcal enterotoxin-E (SEE) and derived from mycoplasma, mycobacterium and the toxin of herpesviral.

Antigen corresponding at least part target antigen can be separated from natural origin, or can use recombination known in the art Technology preparation.For example, can be by the MHC for the antigen presenting cell that the cell mass or tissue for needing to adjust immune response obtain and other It presents molecule and elutes peptide antigen.Can be used standard protein purification technology known in the art purifying elution peptide (Rawson etc., 2000, Cancer Res 60 (16), 4493-4498).If desired, the peptide of purifying can be sequenced, and with as described below Standard protein synthetic technology production synthesis version peptide.Alternatively, can by separate need to adjust immune response cell mass or The sample of tissue, then crack the sample or make the sample be in result in (such as ultraviolet light or γ under conditions of apoptotic cell Ray radiation, virus infection, cell factor or by exhaust the cytotrophy in cell culture medium, with hydrogen peroxide be incubated for, Or with drug such as dexamethasone, ceramide chemotherapeutic and anti-hormone drug such as leuprorelin acetate (Lurpon) or tamoxifen Sweet smell is incubated for), to generate crude antigen preparation.Then, lysate or apoptotic cell can be used as the source of crude antigen, with solvable shape Formula is used or is contacted with antigen presenting cell, is described in further detail below.

In an exemplary embodiment, polypeptide complex of the invention or chimeric polyeptides or the nucleic acid of chimeric polyeptides can be expressed Construct (" immunomodulator ") is used for treating cancer.These embodiments it is some in, immunomodulator can be with inhibition At least one cancer therapy of tumor cell proliferation, survival or vigor is administered simultaneously.Immunomodulator can be after cancer therapy For treating or can be used together before applying therapy or with therapy.Therefore, the present invention considers combination treatment, Using immunomodulator of the invention and cancer therapy is administered simultaneously, non-limiting example include radiotherapy, surgical operation, Chemotherapy, promotees apoptosis therapy and immunotherapy at hormone ablation therapy.

6.2 radiotherapy

Radiotherapy includes radiation and the wave of induced DNA damage, for example, gamma-irradiation, X-ray, UV irradiation, microwave, electricity Sub- transmitting, radioactive isotope etc..Therapy can be realized by irradiating localized tumor site with forms of radiation described above. It is most likely that all of these factors taken together is to the duplication and reparation and the assembling of chromosome of the precursor, DNA of DNA, DNA and dimension It holds and generates broad range of damage.

The dosage range of X-ray continues extended period (3-4 weeks) from the daily dosage of 50-200 roentgen for range, To the single dose of 2000-6000 roentgen.Radioisotopic dosage range variation is very wide, and depends on the half of isotope Decline the phase, transmitting radiation intensity and type and neoplastic cell intake.

The non-limiting example of radiotherapy includes the conformal external beam radiotherapy (50- of single percussion or gradation percussion (fractions) is provided 100 Grays by several times in 4-8 weeks), high dose rate brachytherapy, permanent interstitial closely Radiotherapy, body radioactivity isotope (for example, strontium 89).In some embodiments, radiotherapy can be with radiosensitization Agent is administered in combination.The illustrated examples of radiosensitizer include but is not limited to Efaproxiral (efaproxiral), etanidazole (etanidazole), fluorine road rope (fluosol), Misonidazole (misonidazole), nimorazole (nimorazole), replace Mo Bofen (temoporfin) and Tirapazamine (tirapazamine).

6.3 chemotherapy

Chemotherapeutant can be selected from any one or more of following classification:

(i) antiproliferative agents/antineoplastic and combinations thereof, as used in Medical oncology, such as alkylating agent (such as it is suitable Platinum, carboplatin, cyclophosphamide, mustargen, melphalan (melphalan), Chlorambucil (chlorambucil), busulfan (busulphan) and nitrosoureas), antimetabolite (such as antifol such as fluoropyrimidine class (fluoropyridines), As 5 FU 5 fluorouracil and Tegafur (tegafur), Raltitrexed (raltitrexed), amethopterin (methotrexate), Ah Sugared cytidine and hydroxycarbamide)；Antitumor antibiotics (such as anthracycline, such as adriamycin, bleomycin, Doxorubicin (doxorubicin), daunomycin, epirubicin (epirubicin), idarubicin (idarubicin), Mitomycin-C, Dactinomycin D (dactinomycin) and mithramycin (mithramycin))；Antimitotic agent (such as vinca alkaloids, Such as vincristin (vincristine), vinblastine (vinblastine), eldisine (vindesine) and vinorelbine (vinorelbine) and taxanes such as taxol and docetaxel) and topoisomerase enzyme inhibitor (such as epipodophyllotoxin is such as Etoposide and Teniposide, amsacrine (amsacrine), topotecan and camptothecine)；

(ii) cytostatic agent such as antiestrogenic (such as tamoxifen (tamoxifen), Toremifene (toremifene), Raloxifene (raloxifene), Droloxifene (droloxifene) and Idoxifene (iodoxyfene)), estrogen receptor lowers object (such as fulvestrant (fulvestrant)), antiandrogen (such as than card Shandong Amine (bicalutamide), Flutamide (flutamide), Nilutamide (nilutamide) and Cyproterone Acetate), UH antagonism Agent or LHRH agonist (such as Goserelin (goserelin), Leuprorelin (leuprorelin) and Buserelin (buserelin)), progestational hormone (such as megestrol acetate), aromatase inhibitor are (such as such as Anastrozole (anastrozole), Letrozole (letrozole), Vorozole (vorozole) and Exemestane (exemestane)) and 5 Alpha-reductase inhibitors such as Finasteride；

(iii) inhibit reagent (such as the metal protease inhibitors such as Marimastat of cancer cell invasion (marimastat) and the inhibitor of urokinase-type plasminogen activator function of receptors)；

(iv) growth factor depressant of functions, such as such inhibitor include that growth factor antibodies, growth factor receptors are anti- Body (such as anti-erbb2 antibody trastuzumab [Herceptin^TM] and anti-erbb1 antibody cetuximab [C225]), method Transferase inhibitors, mek inhibitor, tyrosine kinase inhibitor and serine/threonine kinase inhibitor, such as epidermis are raw Other inhibitor (such as other EGFR families tyrosine kinase inhibitor such as N- (the chloro- 4- fluorophenyl of 3-)-of long factor family 7- methoxyl group -6- (3- morpholino propoxyl group) quinazoline -4- amine (Gefitinib (gefitinib), AZD1839), N- (3- acetylene Base phenyl) bis- (2- methoxy ethoxy) quinazoline -4- amine of -6,7- (Tarceva (erlotinib), OSI-774) and 6- third Acrylamide base-N- (the chloro- 4- fluorophenyl of 3-) -7- (3- morpholino propoxyl group) quinazoline -4- amine (CI1033)), such as blood platelet The inhibitor of the inhibitor of the growth factor family in source and such as hepatocyte growth factor family；

(v) those of anti-angiogenic agent, such as inhibition vascular endothelial growth factor effect are (for example, anti-Vascular Endothelial is thin Intracellular growth factor antibody bevacizumab [Avastin^TM], compound is for example in international patent application WO97/22596, WO97/ 30035, those compounds disclosed in WO97/32856 and WO98/13354) and the compound that is acted on by other mechanisms plays (for example, inhibitor and angiostatin of Li Nuoan (linomide), 3 function of beta 2 integrin alpha v β)；

(vi) injury of blood vessel agent, for example, combretastatin A4 and international patent application WO99/02166, WO00/40529, Compound disclosed in WO00/41669, WO01/92224, WO02/04434 and WO02/08213；

(vii) antisense therapy, such as those of target listed above, such as ISIS2503, a kind of anti-ras is anti- Adopted object；And

(viii) gene therapy method, including for example substitute aberrant gene such as exception p53 or exception GDEPT (gene is led To enzyme prodrug treatment) method method, such as using cytosine deaminase, thymidine kinase or bacterium nitroreductase that A bit, and increase patient to the method for the tolerance of chemotherapy or radiotherapy, such as more-drug tolerance gene therapy.

6.4 immunotherapy

The method of immunotherapy includes, such as improves the in vitro and vivo approaches of the immunogenicity of patient tumors cell, such as It is transfected with cell factor (such as interleukin 2, interleukin-4 or granulocyte-macrophage colony stimutaing factor)； The method for reducing T cell anergy；With the method for the immunocyte (dendritic cells of such as cytokine transfection) of transfection；With thin The method of the tumor cell line of intracellular cytokine transfection；And the method with anti-idiotype.These methods often rely on use Immunoeffectors cell and molecule target and destroy cancer cell.It is (such as right that immunoeffectors can be such as antigen binding molecules Marker on tumor cell surface has the antibody of specificity).Individual antigen binding molecules may be used as the effect of therapy Object or its can raise other cells in fact to promote cell killing.Antigen binding molecules can also be with drug or toxin (chemotherapeutant, radionuclide, ricin A chain, cholera toxin, pertussis toxin etc.) conjugation, and it is used only as target To agent.Optionally, immunoeffectors can be lymphocyte, what carrying was directly or indirectly interacted with malignant cell target Surface molecular.Various immunoeffectors cells include cytotoxic T cell and NK cell.

These embodiments it is some in, suitably antigen binding molecules targeting cell surface antigen be tumour correlation Antigen, illustrated examples include Her2/neu, EGFR, Epcam, VEGFR, FGFR, MUC-1, CA 125, CEA, MAGE, The specific antigen of CD20, CD19, CD40, CD33, A3, A33 antibody, BrE3 antigen, CD1, CD1a, CD5, CD8, CD14, CD15、CD16、CD21、CD22、CD23、CD30、CD33、CD37、CD38、CD40、CD45、CD46、CD52、CD54、CD74、 CD79a, CD126, CD138, CD154, B7, Ia, Ii, HM1.24, HLA-DR (for example, HLA-DR10), NCA95, NCA90, HCG With subunit, CEA (CEACAM5), CEACAM-6, CSAp, EGP-1, EGP-2, Ba 733, KC4 antigen, KS-1 antigen, KS1- 4, Le-Y, MUC2, MUC3, MUC4, PlGF, ED-B fibronectin, NCA 66a-d, PAM-4 antigen, PSA, PSMA, RS5, S100, TAG-72, T101, TAG TRAIL-R1, TRAIL-R2, p53, tenascin, insulin growth factor-1 (IGF-I), Tn antigen etc..

6.5 other therapies

The example of other cancer therapies includes light therapy, cold therapy, toxinotherapy or promotees apoptosis therapy.The skill of this field Art personnel will be appreciated by, which is not that can be used for the exhaustion of the therapeutic modality type of cancer and other hyperplastic lesions.

It is well known that chemotherapy and radiation-therapy quickly target dividing cell and/or destroy cell cycle or cell division.This A little treatments are provided as a part for the cancer for treating several forms, it is intended to slow down its progress or inverse by curative therapy Turn the symptom of disease.However, these treatments of cancer may cause immunocompromised host state and subsequent pathogenic infection, and therefore The present invention also extends to combination treatment, and the combination treatment is described using both polypeptide complex, cancer therapy and anti-infective Anti-infective, which is effectively antagonized, to be infected caused by the immunocompromised host situation due to caused by cancer therapy or since cancer therapy causes Immunocompromised host situation and increase the infection of risk.Suitably, anti-infectives are selected from antimicrobial, the antimicrobial The compound of microorganism (such as virus, bacterium, yeast, fungi, protozoan etc.) growth is including but not limited to killed or inhibits, It and therefore include antibiotic, amebicide, antifungal agent, antiprotozoal agent, antimalarial, anti-tubercular drug and antiviral agent.It is anti- Infection medicine also includes antihelmintic (anthelmintics) and nematicide within its scope.Illustrative antibiotic includes quinoline Promise ketone (quinolones) is (for example, Amifloxacin (amifloxacin), cinoxacin (cinoxacin), Ciprofloxacin (ciprofloxacin), Enoxacin (enoxacin), fleraxacin (fleroxacin), flumequine (flumequine), Lip river Beautiful Sha Xing (lomefloxacin), acidum nalidixicum (nalidixic acid), Norfloxacin (norfloxacin), Ofloxacin (ofloxacin), lavo-ofloxacin (levofloxacin), Lomefloxacin, oxolinic acid (oxolinic acid), pefloxacin (pefloxacin), Rosoxacin (rosoxacin), Temafloxacin (temafloxacin), Tosufloxacin (tosufloxacin), Sparfloxacin (sparfloxacin), Clinafloxacin (clinafloxacin), gatifloxacin (gatifloxacin), Moxifloxacin (moxifloxacin)；Gemifloxacin (gemifloxacin)；And T-3811 (garenoxacin)), tetracycline (tetracyclines), sweet ammonia ring plain (glycylcyclines) and oxazolidone (oxazolidinones) (for example, aureomycin (chlortetracycline), demeclocycline (demeclocycline), mostly west Ring element (doxycycline), lymecycline (lymecycline), metacycline (methacycline), minocycline (minocycline), oxytetracycline (oxytetracycline), tetracycline (tetracycline), tigecycline (tigecycline)；Linezolid (linezolide), eperezolid (eperezolid)), glycopeptide (glycopeptides), Aminoglycoside (aminoglycosides) is (for example, amikacin (amikacin), Arbekacin (arbekacin), fourth glycosides bacterium Plain (butirosin), dibekacin (dibekacin), fortimicins (fortimicins), gentamicin (gentamicin), Kanamycins (kanamycin), neomycin, Netilmicin (netilmicin), ribostamycin (ribostamycin), Xi Suo meter Star (sisomicin), spectinomycin (spectinomycin), streptomysin (streptomycin), tobramycin (tobramycin)), beta-lactam is (for example, Imipenem (imipenem), Meropenem (meropenem), Biapenem (biapenem), Cefaclor (cefaclor), cefadroxil (cefadroxil), cefadole (cefamandole), head Spore Qu Qin (cefatrizine), Cefazedone (cefazedone), cephazoline (cefazolin), Cefixime (cefixime), Cefmenoxime (cefmenoxime), cefodizime (cefodizime), cefonicid (cefonicid), head Spore piperazine ketone (cefoperazone), ceforanide (ceforanide), cefotaxime (cefotaxime), Cefotiam (cefotiam), Cefpimizole (cefpimizole), cefpiramide (cefpiramide), Cefpodoxime (cefpodoxime), Cefsulodin (cefsulodin), cefotaxime (ceftazidime), Cefteram (cefteram), ceftezole (ceftezole), Ceftibuten (ceftibuten), Ceftizoxime (ceftizoxime), ceftriaxone (ceftriaxone), Cefuroxime (cefuroxime), Cefuzonam (cefuzonam), cephaacetrile, cefalexin (cephalexin), Cefaloglycin (cephaloglycin), cefaloridine (cephaloridine), cefoxitin (cephalothin), cephalo Woods (cephapirin), Cefradine (cephradine), cefmetazole (cefinetazole), Cefoxitin (cefoxitin), cefotetan (cefotetan), aztreonam (azthreonam), carumonan (carumonam), fluorine oxygen head Spore (flomoxef), latamoxef (moxalactam), Mecillinam (amidinocillin), Amoxicillin (amoxicillin), ampicillin (ampicillin), azlocillin (azlocillin), carbenicillin (carbenicillin), parasiticin (benzylpenicillin), carfecillin (carfecillin), Cloxacillin (cloxacillin), dicloxacillin (dicloxacillin), methicillin (methicillin), mezlocillin (mezlocillin), naphthlazole (nafcillin), oxacillin (oxacillin), benzyl penicillin (penicillinG), piperazine Draw XiLin (piperacillin), sulbenicillin (sulbenicillin), temocillin (temocillin), Ticarcillin (ticarcillin), Cefditoren (cefditoren), SC004, KY-020, Cefdinir (cefdinir), Ceftibuten (ceftibuten), FK-312, S-1090, CP-0467, BK-218, FK-037, DQ-2556, FK-518, Cefozopran (cefozopran), ME1228, KP-736, CP-6232, Ro 09-1227, OPC-20000, LY206763), rifamycin (rifamycins), macrolide (macrolides) (such as azithromycin (azithromycin), clarithromycin (clarithromycin), erythromycin (erythromycin), oleandomycin (oleandomycin), rokitamycin (rokitamycin), rosamicin (rosaramicin), roxithromycin (roxithromycin), troleandomycin (troleandomycin)), (such as Ketek (telithromycin), quinoline are red mould for ketone lactone antibiotic (ketolides) Plain (cethromycin)), coumermycin (coumermycins), lincosamide antibiotic (lincosamides) (for example, gram Woods mycin (clindamycin), lincomycin (lincomycin) and chloramphenicol (chloramphenicol)).

Illustrative antiviral agent includes abacavir sulfate (abacavir sulfate), acyclovir sodium (acyclovir Sodium), amantadine hydrochloride (amantadine hydrochloride), anpunave (amprenavir), cidofovir (cidofovir), delavirdine mesilate (delavirdine mesylate), Didanosine (didanosine), Yi Feiwei Human relations (efavirenz), famciclovir (famciclovir), Fomivirsen sodium (fomivirsen sodium), Foscarnet sodium (foscarnet sodium), Ganciclovir (ganciclovir), indinavir sulfate (indinavir sulfate), rummy Husband determines (lamivudine), Lamivudine/Zidovudine (zidovudine), nelfinavir mesilate (nelfinavir Mesylate), nevirapine (nevirapine), Oseltamivir phosphate (oseltamivir phosphate), Ribavirin (ribavirin), rimantadine hydrochloride (rimantadine hydrochloride), Ritonavir (ritonavir), Sha Kui That Wei (saquinavir), saquinavir mesilate (saquinavir mesylate), stavudine (stavudine), hydrochloric acid Valaciclovir (valacyclovir hydrochloride), zalcitabine (zalcitabine), zanamivir (zanamivir) and Zidovudine.

The non-limiting example of amebicide or antiprotozoal agent includes Atovaquone (atovaquone), chloroquine hydrochloride (chloroquine hydrochloride), chloroquine diphosphate (chloroquine phosphate), metronidazole (metronidazole), hydrochloric acid metronidazole (metronidazole hydrochloride) and pentamidine isethionate (pentamidine isethionate).Antihelmintic can be at least one selected from the following: mebendazol (mebendazole), Pyrantel Pamoate (pyrantel pamoate), albendazole (albendazole), ivermectin (ivermectin) and thiabendazolum (thiabendazole).Illustrative antifungal agent can be selected from amphotericin B (amphotericin B), amphotericin B Cholesterol sulfate ester complexes (amphotericin B cholesteryl Sulfate complex), amphotericin B lipid complex (amphotericin B lipid complex), amphotericin B Liposome (amphotericin B liposomal), Fluconazole (fluconazole), Flucytosine (flucytosine), ash Flavomycoin particle (griseofulvin microsize), griseofulvin ultramicrosize (griseofulvin Ultramicrosize), Itraconazole (itraconazole), ketoconazole (ketoconazole), nystatin (nystatin) and terbinafine HCl (terbinafine hydrochloride).The non-limiting example of antimalarial includes Chloroquine hydrochloride (chloroquine hydrochloride), chloroquine diphosphate (chloroquine phosphate), Doxycycline, Hydroxychloroquine sulfate (hydroxychloroquine sulfate), Mefloquine Hydrochloride (mefloquine Hydrochloride), primaquine phosphate (primaquine phosphate), pyrimethamine (pyrimethamine) and second Amic metadiazine-sulfadoxine (pyrimethamine with sulfadoxine).Antituberculotic includes but is not limited to chlorine Fawzi Bright (clofazimine), seromycin (cycloserine), dapsone (dapsone), ebutol (ethambutol Hydrochloride), isoniazid (isoniazid), pyrazinamide (pyrazinamide), Mycobutin (rifabutin), Rifampin (rifampin), Rifapentine (rifapentine) and streptomycin sulphate (streptomycin sulfate).

In other embodiments that Th1 related disease is pathogenic infection, polypeptide complex of the invention with it is anti-infective Agent is administered simultaneously, such as described above.

As noted above, it is total together with additional or complementary medicament to cover immunomodulator of the invention by the present invention Application.It will be understood that in the embodiment for including application immunomodulator and other one or more medicaments, active matter in combination The dosage of matter can include effective quantity with itself, and additional medicament can further increase the benefit of the treatment or prevention to patient Place.Optionally, immunomodulator and other medicament can include for preventing or treating Th1 related disease or illness together Effective quantity.It will also be understood that can give a definition effective quantity in the background of particular treatment, particular treatment includes, such as applies Time and number, method of application, preparation etc..In some embodiments, immunomodulator and optionally cancer therapy is pressed Routine schedule application.It is alternatively possible to apply cancer therapy when symptom occurs." routine schedule " refers to as used herein Scheduled designated time period.As long as timetable be it is scheduled, routine schedule can cover the period of identical or different length. For example, routine schedule may include daily, every two days, every three days, it is four days every, five days every, six days every, weekly, monthly or the phase Between any setting number of days or all numbers, the every two moon, three months, four months, five months, six months, seven months, eight months, The application polypeptide complex such as nine months, ten months, 11 months, 12 months.Optionally, scheduled routine schedule can wrap Include first week and polypeptide complex and cancer therapy be administered simultaneously daily, subsequent periods of months is monthly applied, then herein it Every three months is applied afterwards.As long as it includes the application at specific certain day that reasonable time table, which is determined in advance, routine schedule is covered Any specific combination.

6.6 dosage and administration method

Composition is applied with " effective quantity ", i.e., effectively realizes the amount of expected purpose in subject.It is applied to the work of patient Property compound dosage should be enough in subject to realize at any time beneficial to reaction, such as reduce relevant to Th1 disease or disease The relevant at least one symptom of disease.The amount or dose frequency of pharmaceutical active compounds to be administered may depend on to be treated tested Person, including its age, gender, weight and general health.It in this respect, will for the precise volume of the reactive compound of application Judgement depending on operator.By routine experiment, those skilled in the art can determine immunomodulator as described herein Effectively, nontoxic amount, to include obtaining required treatment results in pharmaceutical composition of the invention.

In general, pharmaceutical composition of the invention can be according to physical trait (including the health with administration method and recipient State) compatible mode is applied, and application causes required effect (that is, treatment validity, immunogenicity in this way And/or protectiveness).For example, the suitable dose of pharmaceutical composition of the present invention may depend on many factors, it is including but not limited to tested The physical trait (for example, age, weight, gender) of person；Whether this compound is used as single medicament or complementary therapy；Patient's MHC Limit Type；The progress (i.e. pathological state) of virus infection；And it will be appreciated by the appropriately skilled person that other because Element.When determining the suitable dose of pharmaceutical composition of the present invention, it may be considered that various general Considerations be described in for example Gennaro (2000) " Remington:The Science and Practice of Pharmacy ", the 20th edition, Lippincott, Williams , &Wilkins；With Gilman et al. (eds.) (1990) " Goodman and Gilman's:The Pharmacological Bases of Therapeutics ", Pergamon Press.

In some embodiments, " effective quantity " of subject immune's regulator is to be enough to realize desired prevention or treatment The amount of effect, such as to reduce disease relevant to Th1 or the relevant symptom of illness.In these embodiments, exempt from unused The individual symptom of epidemic disease modulators for treatment is compared, and effective quantity subtracts disease relevant to Th1 or the relevant symptom of illness in individual Few at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more.The symptom of various Th1 related diseases or illness and measure these diseases The method of shape is known in the art.For example, for measuring tumor load, tumor grade, pathogenic organism quantity etc. in individual Method be this field Plays.

In some embodiments, " effective quantity " of subject immune's regulator is effectively drawn in the administration method of selection Send out the amount of immune response (including Th1 immune response).The method for measuring immune response (including Th1 immune response) is that this field is general Known to logical technical staff.Illustrative methods include that solid phase heterogeneous assays (for example, enzyme linked immunosorbent assay (ELISA)), solution are mutually surveyed Fixed (for example, electrochemical luminescence measurement), the Luminescent Proximity Homogenous measurement of amplification, flow cytometry, intracellular cytokine dye, Functional T cell measurement, functional B cell measurement, functional Monocyte-macrophages measurement, dendron and reticular endothelium are thin Viral RNA/DNA determines in IFN-γ, tissue or the biofluid that born of the same parents' measurement, the measurement of NK cell response, immunocyte generate Amount (for example, in serum or other fluids or tissue/organ viral RNA or DNA quantify), oxidative burst measurement, cytotoxicity- Specific cell lysis measurement, pentamer binding assay and phagocytosis and Apoptosis assessment.

Pharmaceutical composition of the invention can be applied to recipient by standard way, including but not limited to parenteral (for example, Intravenously).

Pharmaceutical composition of the invention can be applied to recipient discretely or with other therapeutic agent.In medicine group It closes in the embodiment that object and therapeutic agent are administered simultaneously, application, which can be, simultaneously or sequence (applies pharmaceutical composition, so After apply medicament, vice versa).

In general, in treatment use, lasting period that treatment can be used for during morbid state or illness.In addition, for this Field those of ordinary skill is it is readily apparent that by by the shape of the property and degree of morbid state to be treated or illness, application The property of formula, approach and position and particular individual to be treated determines optimised quantity and the interval of individual dose.It can be used often Rule technology determines optimum condition.

(for example, prophylactic use) in many cases, it may be necessary to several times or multiple applications medicine group of the invention Close object.For example, pharmaceutical composition can be applied 1,2,3,4,5,6,7,8,9,10 or more times.Application can be about 1 to about 12 The interval in week, and in certain embodiments, it can be about 1 to about 4 week interval.It is being repeated exposure to by drug of the present invention In the case where special pathogen or other diseases Related Component that composition is targeted, it may be necessary to periodically apply again.

It will be apparent to those skilled in the art that the conventional mistake that treatment determines test can be used Journey, to determine optimal application process.

When two or more entities " joint " or when being applied to subject " simultaneously ", they can be simultaneously with single combination Object application, or be administered simultaneously in individual composition, or applied in single formulation separated in time.

In certain embodiments, the present invention relates to multiple individually dosed application pharmaceutical compositions.Therefore, herein for pre- Prevent and treat Th1 related disease or the method for illness includes, such as is interior multiple separated to subject's application in a limited time period Dosage.Therefore, the method disclosed herein for preventing and treating infection includes applying the pharmaceutical composition of the invention of amount of initiator Object.It can be booster after amount of initiator.Booster can be used for inoculating.In various embodiments, medicine group Close object or vaccine administration at least once, twice, three times or more.

7. treatment method

Immunomodulator of the invention, which can be used for treating, reduces or is damaged relevant disease to Th1 immune response.For example, Th1 related disease may be virus, bacterium, fungi or parasitic infection.Virus includes but is not limited to that Retroviridae people exempts from Epidemic disease defective virus, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV or HIV-III)；With other separation strains (such as HIV-LP)；Picornaviridae (Picornaviridae) (such as poliovirus, hepatitis A disease Poison, enterovirus, human Coxsackie virus, rhinovirus, echovirus), Caliciviridae (Calciviridae) (such as causes The strain of gastroenteritis, including Cécile Nowak and correlated virus)；Togaviridae (Togaviridae) (such as equine encephalomyelitis virus, Rubella virus)；Flaviviridae (Flaviviridae) (such as dengue fever virus, encephalitis viruses, yellow fever virus)；Coronaviridae (Coronoviridae) (such as coronavirus)；Rhabdoviridae (Rhabdoviridae) (for example, vesicular stomatitis virus, Rabies viruses)；Filamentous virus section (Filoviridae) (such as Ebola (ebola) virus), Paramyxoviridae (Paramyxoviridae) (such as parainfluenza virus, mumps virus, measles virus, Respiratory Syncytial Virus(RSV), metaneumovirus)；Orthomyxoviridae family (Orthomyxoviridae) (for example, influenza virus)；Bunyaviridae (Bungaviridae) (such as Hantaan virus (Hataanvirus), bunga virus, sand fly viral (phleoboviruses) and Nairo virus)；Arenavirus section (Arenaviridae) (hemorrhagic fever viruse)；Reoviridae (Reoviridae) (such as Reovirus, Orbivirus and rotavirus)；Infectious bursa of Fabricius virus category birnavirus section (Bimaviridae)；Liver Nuclifort Viraceae (Hepadnaviridae) (hepatitis type B virus)；Parvoviridae (Parvoviridae) is (thin Small virus)；Papovaviridae (Papovaviridae) (papillomavirus, polyomavirus)；Adenoviridae (Adenoviridae) (most adenovirus), herpetoviridae (Herpeviridae) (herpes simplex virus (HSV) 1 and 2, water Acne herpes zoster virus, cytomegalovirus (CMV), herpesviral)；Poxviridae (Poxyiridae) (variola virus, VACV, Poxvirus)；With iris Viraceae (Iridoviridae) (for example, African swine fever virus)；With non-classified virus (for example, sea The causative agent of continuous shape encephalopathy, the causative agent of hepatitis D (being considered as the deficiency satellite virus of hepatitis type B virus), non-first, Causative agent (1 class=intestinal transmitted of non-hepatitis B；(i.e. the hepatitis C) that 2 classes=parenteral is propagated)；And astrovirus.

In some embodiments, pathogenic infection is bacterial pathogens.Being known to be the pathogenic bacterium of subject includes, But pathogenicity pasteurella (Pasteurella) species are not limited to (for example, multocida (Pasteurella Multocida)), staphylococcus (Staphylococci) species are (for example, staphylococcus aureus (Staphylococcus Aureus)), streptococcus (Streptococcus) species are (for example, streptococcus pyogenes (Streptococcus pyogenes) (A group of streptococcus), Streptococcusagalactiae (Streptococcus agalactiae) (B group of streptococcus), (green group of streptococcus (viridans group)), streptococcus fecalis (Streptococcus faecalis), bargen's streptococcus (Streptococcus Bovis), streptococcus (anaerobism kind (anaerobic sps.)), streptococcus pneumonia (Streptococcus Pneumoniae)), eisseria (Neisseria) species are (for example, Diplococcus gonorrhoeae (Neisseria Gonorrhoeae), Neisseria meningitidis (Neisseria meningitides)), Escherichia (Escherichia) species are (for example, enterotoxigenic Escherichia coli (E.coli) (ETEC), cause enteropathogenic escherichia (EPEC), intestines Enterohemorrhagic E.coli (EHEC) and enteroinvasive E coli (EIEC)), Bordetella (Bordetella) species, Campylobacter (Campylobacter) species, Legionnella (Legionella) species are (for example, invade lung Legionella (Legionella pneumophila)), pseudomonas (Pseudomonas) species, Shigella (Shigella) object Kind, vibrio (Vibrio) species, Yersinia (Yersinia) species, Salmonella (Salmonella) species, Haemophilus spp (Haemophilus) species (for example, Hemophilus influenzae (Haemophilus influenzae)), Brucella (Brucella) species, Francisella category (Francisella) species, Bacteroides (Bacterioides) species, shuttle Pseudomonas (Clostridia) species are (for example, clostridium difficile (Clostridium difficile), C.perfringens (Clostridium perfringens), clostridium tetani (Clostridium tetani)), Mycobacterium (Mycobacteria) species are (such as mycobacterium tuberculosis (M.tuberculosis), mycobacterium avium (M.avium), intracellular Mycobacteria (M.intracellulare), mycobacterium kansasii (M.kansaii), mycobacterium gordonae (M.gordonae)), helicobacter pylori (Helicobacter pyloris), B. burgdorferi (Borelia Burgdorferi), monocyte Listeria monocytogenes (Listeria monocytogenes), chlamydia trachomatis (Chlamydia trachomatis), enterococcus spp (Enterococcus) species, Bacillus anthracis (Bacillus Anthracis), corynebacterium diphtheriae (Corynebacterium diphtheriae), erysipelothrix rhusiopathiae (Erysipelothrix rhusiopathiae), clostridium perfringen (Enterobacter aerogenes), kerekou pneumonia primary Salmonella (Klebsiella pneumoniae), Fusobacterium nucleatum (Fusobacterium nucleatum), Streptobacillus moniliformis (Streptobacillus moniliformis), Treponema pallidum (Treponema palladium), superfine treponema (Treponema pertenue), Leptospira (Leptospira), rickettsia (Rickettsia) and Israel Actinomyces (Actinomyces israeli).

In other embodiments of the present invention, pathogenic infection is eukaryotic pathogens, such as pathogenic epiphyte and parasitism Worm.It is known to be pathogenic fungi and includes, but are not limited to Cryptococcus neoformans (Cryptococcus at least to a certain extent Neoformans), histoplasma capsulatum (Histoplasma capsulatum), posadasis spheriforme (Coccidioides Immitis), Blastomyces dermatitidis (Blastomyces dermatitidis), candida albicans (Candida albicans), smooth Candida albicans (Candida glabrata), aspergillus fumigatus (Aspergillus fumigate), aspergillus flavus (Aspergillus ) and Sporothrix schenckii (Sporothrix schenckii) flavus.

Other eukaryotic pathogens that heterologous antigen can be derived include, but are not limited to causative protozoa, worm (helminth), plasmodium (Plasmodium), such as plasmodium falciparum (Plasmodium falciparum), malarlae malaria are former Worm (Plasmodium malariae), Plasmodium ovale (Plasmodium ovale) and Plasmodium vivax (Plasmodium vivax)；Toxoplasma gondii (Toxoplasma gondii)；Trypanosoma bocagei (Trypanosoma brucei), schizotrypanum cruzi (Trypanosoma cruzi)；Schistosoma haematobium (Schistosoma haematobium), schistosoma mansoni (Schistosoma mansoni), Schistosoma japonicum (Schistosoma japonicum)；Leishmania donovani (Leishmania donovani)；Giardia intestinalis (Giardia intestinalis)；Cryptosporidium parvum (Cryptosporidium parvum)；Deng.

May other diseases relevant to reduction or impaired Th1 immune response further include disease before any pernicious or canceration Disease, proliferative or hyperproliferative disorders or due to proliferative capacity or any cell or tissue behavior of body in terms of functionality Or derivative or relevant any disease caused by other interference or exception.Polypeptide complex and group of the invention can be used The non-limiting cancer for closing object treatment includes the cancer (packet of breast cancer, colon cancer, lung cancer and prostate cancer, blood and lymphatic system Include Hodgkin's disease, leukaemia, lymthoma, Huppert's disease and waldenstrom's disease), cutaneum carcinoma (including maligna Plain tumor), alimentary tract cancer (including head and neck cancer, cancer of the esophagus, gastric cancer, cancer of pancreas, liver cancer, colorectal cancer, cancer of anus), genitals With urinary system cancer (including kidney, bladder cancer, carcinoma of testis, prostate cancer), female cancer (including breast cancer, oophoroma, gynaecology Cancer and choriocarcinoma) and brain, bone benign tumour, nasopharyngeal carcinoma, peritonaeum after cancer, thyroid cancer and soft tissue neoplasm.

8.Th1 immune state biomarker and application thereof

The present invention is also based partially on determining PD-L2 and interacts on cell (including APC, such as dendritic cells) in IEC- Expression, this kind expression is negatively correlated with the severity of Th1 related disease, and PD-L2 is to establish the immune necessary condition of Th1.Cause This, present inventor have determined that PD-L2 is the dependable indicator for the Th1 immune response for raising and/or enhancing in subject.They The other biomarkers object adjusted during Th1 immune response is also found.These additional biomarkers are included in increase The diagnosis capability and reliability of diagnosis teaching herein and prognosis measurement.Based on these determination, propose PD-L2 (optionally and its He combines Th1 immune state biomarker) indicate the Th1 immune state of subject, and Th1 correlation disease is suffered from for tracking The development of Th1 immune state in the subject of disease.

Therefore, the adjoint diagnosis as polypeptide complex of the present invention and chimeric polyeptides, the present invention also provides for identifying Subject Th1 immune state or method, apparatus for providing prognosis for the subject with Th1 related disease, composition and Kit.

8.1 Th1 immune state biomarkers

Present inventor have determined that certain surface markers are present on IEC- interaction cell, including for example dendron is thin The APC of born of the same parents, it is specific expressed in people and mouse during Th1 immune response.Result provided herein provides explicitly Evidence, i.e., unique biology correlation biomarker spectrum is with the Th1 immune state of high accuracy prediction subject.It is overall and Speech, these discoveries provide compellent evidence, it was demonstrated that IEC- interaction cell surface marker object disclosed herein is (special It is not PD-L2) it can be used as biomarker for determining Th1 immune state, and can be potentially with making to subject Classification treatment decision useful diagnosis, wherein the subject suffer from disease relevant to undesirable Th1 immune state.? This respect proposes that method disclosed herein, device, composition and kit based on these biomarkers can be used for pinpointing photograph The diagnosis of shield, and allow quickly and inexpensively to determine Th1 immune state, therefore the cost of medical system can be saved significantly on, this is It is suitable for enhancing or eliminating Th1 in subject if necessary because the subject with undesirable Th1 immune response can be exposed to The therapeutic agent of immune response.

Using method described herein, some biomarkers are identified, for determining that the Th1 of subject is immune State is particularly useful.These biomarkers are known as " Th1 immune state biomarker ".As used herein, " Th1 exempts from term Epidemic disease state biomarker " refers to the biomarker of subject, the usually biomarker of subject immune system, makees For Th1 immune response a part and be changed or its expression is changed.Th1 immune state biomarker is gene Appropriate expression product (being also interchangeably referred to as " Th1 immune response biomarker gene " herein), including polynucleotides and Polypeptide expression products.As used herein, the polynucleotides expression product of Th1 immune state biomarker gene is herein referred to as " Th1 immune state biomarker polynucleotides ".Polypeptide expression products this paper quilt of Th1 immune response biomarker gene Referred to as " Th1 immune state biomarker polypeptide ".

Suitably, at least one Th1 immune state biomarker of the invention includes PD-L2.Natural human PD-L2 amino Acid sequence is as shown in SEQ ID NO:1, and by nucleic acid sequence encoding:

Another Th1 immune state biomarker that can be optionally used in the method for the present invention is PD-L1.Natural human PD-L1 amino acid sequence is:

And by nucleic acid sequence encoding:

In above-mentioned Th1 immune state biomarker, it has been found that PD-L2 polypeptide itself has strong diagnosis performance, uses In detection Th1 immune state (for example, measuring PD-L2 using facs analysis⁺The percentage of IEC- interaction cell (such as APC) And measure).Therefore, in specific embodiments, PD-L2 biomarker can be used alone or be immunized with other Th1 State biomarker is combined for determining indicant.Suitably, in these embodiments, biomarker value is to be directed to PD-L2 biomarker and another optional Th1 immune state biomarker (for example, PD-L1) are measured or derive, with Determine indicant.

The present inventor also determines, when being applied in combination with PD-L2 biomarker, other Th1 immune state biomarkers Object has strong diagnosis performance.In advantageous embodiment, identifies and can be used for determining that the Th1 immune state of indicant is raw Pair of substance markers object.Therefore, in such representative example, and as described in detail later, determine indicant with The ratio of Th1 immune state biomarker is related, and ratio can be used for determining the Th1 immune state of subject.

Therefore, the specific proteins product as Th1 immune response biomarker is disclosed herein, provides for determining The means of the Th1 immune state of subject.By analyzing them in subject or from the level in the sample that subject obtains, comment Estimate these Th1 immune state biomarkers, the biomarker value of measurement or derivation be provided, for determine can be used for assess by The indicant of Th1 immune state in examination person.

8.2 sample preparation

In general, in Th1 immune state biomarker analyte detection or before quantifying, processed sample.For example, can be in analysis The preceding extracting and developing from sample and/or purifying protein and/or nucleic acid.Various albumen, DNA and/or mRNA are extracted and purification technique It is well known to those skilled in the art.Processing may include centrifugation, ultracentrifugation, ethanol precipitation, filtering, fractionation, resuspension, dilute It releases, be concentrated.In some embodiments, the method instructed above and elsewhere provide from primary sample (for example, Biofluid such as blood, serum etc.) analysis (for example, protein biomarkers object quantifies), without processing or limited processing.

Furthermore, it is possible to Th1 immune state biomarker analyte detection or it is quantitative before processed sample, to purify or be enriched with sample The specific part of product or interested cell type.For example, the cell that can be interacted with the IEC- in enriched sample, such as APC Or the specific subgroup (for example, dendritic cells, macrophage, monocyte, B cell or combinations thereof) of tumour cell or APC.? In preferred embodiment, Th1 immune state biomarker analyte detection or it is quantitative before, the IEC- interaction in enriched sample Cell, such as APC is (for example, dendritic cells, including CD11c⁺Dendritic cells) and tumour cell.For being enriched with specific cells class The method of the biological sample of type is well known in the art.It is, for example, possible to use such as Ficoll-Hypaque or for cell point From Sucrose gradient solutions, dendritic cells are separated by difference gradient separations, then to remaining pollution red blood cell carry out chlorine Change ammonium or hypotonic lysis (" Cell Biology:A Laboratory Handbook ", Volumes I-III Cellis, J.E.,ed.(1994)；"Current Protocols in Immunology"Volumes I-III Coligan J.E., ed.(1994)；Stites et al. (eds.)).

Sample preparation methods may include step: homogenised sample, removal pollutant and/or measurement in suitable buffer Inhibitor, addition Th1 immune state biomarker capture reagent are (for example, raw with that can specifically bind target Th1 immune state Substance markers object part connection magnetic bead), promote target organisms marker with capture reagent in conjunction under conditions of incubation with life At target organisms marker: capturing the compound of reagent and be incubated for target organisms under the release conditions of target organisms marker Marker: the compound of capture.In some embodiments, by the way that multiple Th1 immune state biomarkers are added into solution Object captures reagent (for example, being specific to required biomarker), separates multiple Th1 immune state biomarkers in the separation of every wheel Object.For example, biomarker is nucleic acid in illustrative embodiment, multiple Th1 immune state biomarkers capture reagent The oligonucleotides for being specific to different target Th1 immune state biomarkers is respectively contained, the capture reagent is added to sample For separating multiple Th1 immune state biomarkers in product.It is expected that this method includes multiple experimental designs, in capture step Number and the quantitative aspects of target Th1 immune state biomarker that is captured in each capture step be varied. In some embodiments, capture reagent be preferential (for example, specifically and selectively) and to be separated, purifying, detection and/ Or molecule, part, substance or the composition of quantitative biomarker-specific object interaction.With with specific Th1 immune state The required binding affinity of biomarker and/or any capture reagent of specificity are used equally for this technology.For example, capture examination Agent can be macromolecular such as peptide, albumen (for example, antibody or receptor), oligonucleotides, nucleic acid (for example, shape can be immunized with Th1 The nucleic acid of state biomarker hybridization), vitamin, oligosaccharides, carbohydrate, lipid or small molecule or its compound.As saying Bright property and non-limiting example, avidin target capture reagent can be used for separating and purifying the target comprising biotin moiety Mark, antibody can be used for separating and purifying the target comprising appropriate antigen or epitope, and oligonucleotides can be used for separating and purifying Complementary oligonucleotides.

Any nucleic acid that can combine or specifically bind target Th1 immune state biomarker is (including single-stranded and double Chain nucleic acid) it can be used as capturing reagent.The example of such nucleic acid includes DNA, RNA, aptamer, peptide nucleic acid and to sugar, phosphoric acid or core Other modifications of glycosides base.Accordingly, there exist many acquisition target target strategies, therefore many types known to those skilled in the art Capture reagent.

In addition, Th1 immune state biomarker capture reagent may include the capture reagent such as positioning, concentration, aggregation Function, when the capture of captured reagent (such as combination, hybridization etc.) (for example, when forming target: when capturing the compound of reagent), To provide separation and purify the mode of target Th1 immune state biomarker.For example, in some embodiments, capture The part (for example, polypeptide) and allow by user on a macroscopic scale that reagent and Th1 immune state biomarker interact The solid support (for example, pearl, surface, resin, column etc.) of operation connects.In general, solid support allows using mechanical means It is separated from heterogeneous solution and purifies target: capture reagent complex.For example, when being connect with pearl, by from heterogeneous molten Pearl (such as passing through physical motion) is removed in liquid realizes separation.It is in magnetic or paramagnetic embodiment in pearl, magnetic field is used In the physical separation for realizing capture reagent (therefore with target Th1 immune state biomarker) with heterogeneous solution.

The assessment of 8.3 Th1 immune state biomarker polypeptides

In order to obtain biomarker value, any suitable technology known in the art can be used to quantify or detect Th1 immune state biomarker.In specific embodiments, using determining individual Th1 immune state biomarker The reagent of horizontal, abundance or amount quantifies Th1 immune state biomarker.Such non-limiting reagent includes being used for The reagent of measurement based on albumen and based on nucleic acid.

By proving the presence of albumen or by one or more known function characteristics of biomarker, in albumen table The expression of Th1 immune state biomarker is assessed on up to level.For example, the anti-PD-L2 for the detection of PD-L2- specific proteins Antibody is described in U.S. Patent number 7,709,214；U.S. Patent application No. Ser. 2009/296,392；And european patent number 1537878, entire contents are incorporated herein by reference.Antibody combines the PD-L2 albumen naturally and being denaturalized, and can be by this Several well known measuring methods in field detect, including enzyme linked immunosorbent assay (ELISA) (ELISA), radiommunoassay (RIA), Luminescent immunoassay, western blot analysis, immunofluorescence assay, immunohistochemistry and fluorescence activated cell sorts (FACS) analysis.

ELISA and RIA follows the similar principles for detecting specific antigen.As an illustrative example, PD-L2 can be with It (is usually used by radioactive label¹²⁵I PD-L2 specific antibody) is measured using RIA.In ELISA measurement, PD-L2 Specific antibody is connect with enzymology.PD-L2 specificity capture antibody is fixed on solid support.It then will be unlabelled Sample, such as carry out the protein extract of biological sample and immobilized antibody is incubated under conditions of non-specific binding is blocked It educates, and removes unbonded antibody and/or albumen by washing.It is combined by the antibody test of the 2nd PD-L2 specific marker PD-L2.Antibody combination is measured directly in RIA by measuring radioactivity, and in ELISA, by the way that colorless substrate is converted It is combined for the reaction of colored reaction product to detect, the colored reaction product is as the active function of ligase.Therefore can lead to It crosses spectrophotometry and easily detects variation (Janeway C.A. et al. (1997) " Immunobiology " third edition supplement Current Biology Ltd.,Garland Publishing Inc.；" Cell Biology:A Laboratory Handbook ", rolls up I-III Cellis, and J.E compiles (1994)；" Current Protocols in Immunology " rolls up I-III Coligan J.E. compiles (1994)；Stites et al. (eds.)).Therefore, two kinds of measurements both provide PD-L2 albumen in biological sample The quantitative approach of content.

The expression of protein biomarkers object can also be detected by luminescence immunoassay.It is closely similar with ELISA and RIA, In luminescent immunoassay, biological sample/protein extract to be tested is fixed on solid support, and is marked with specificity Note, the anti-PD-L2 antibody marked are detected.In turn, label is luminous, and is shone when combining, and is known as specificity Other instruction.Luminescent marking includes the substance luminous when being activated by electromagnetic radiation, electrochemistry excitation or chemical activation, and It may include fluorescence and phosphorus, scintillator and chemiluminescent substance.Label can be a part of catalytic reaction system, such as Enzyme, enzyme fragment, zymolyte, enzyme inhibitor, coenzyme or catalyst；A part of chromogen system, such as fluorogen, dyestuff, chemistry hair Photo etching, luminous agent or sensitizer；It is non magnetic or magnetic dispersible particle, solid support, liposome, ligand, receptor, half anti- Primary emission isotope etc. (U.S. Patent number 6,410,696, U.S. Patent number 4,652,533 and European Patent Application No. 0, 345,776), and additional high-sensitivity method for detecting PD-L2 protein expression is provided.

Western blot analysis is the another kind side for assessing Th1 immune state biomarker content of peptides in biological sample Method.The protein extract of biological sample from IEC- interaction cell (such as APC (for example, dendritic cells) or tumour cell) It is dissolved in denaturation ionization environment, and aliquot is applied to polyacrylamide gel matrix.When albumen is migrated to anode, Based on molecular dimension property protein isolate.Then antigen is transferred on nitrocellulose filter, PVDF or nylon membrane, is then carried out Film is closed so that non-specific binding minimizes.With the antibody detection membrane directly with detectable part coupling, or then with containing The secondary antibody detection membrane of detectable part.In general, horseradish peroxidase or alkaline phosphatase and antibody coupling, color development or luminous bottom Object is for showing activity (Harlow E. et al., (1998) Immunoblotting.In Antibodies:A Laboratory Manual, pp.471-510CSH Laboratory, Cold Spring Harbor, N.Y and Bronstein I. et al. (1992) Biotechniques 12:748-753).

With RIA, ELISA, luminescent immunoassay and the immunoblotting of protein biomarkers object content in quantitatively entire sample Difference, immunofluorescence/immunocytochemistry, still can be according to cell-specific manners for examining although compromising at quantitative aspect Survey albumen.

As set forth above, it is possible to which IEC- interaction cell, such as APC are separated or are enriched with by methods known in the art (for example, dendritic cells) or tumour cell.The separation or enrichment of IEC- interaction cell refer to wherein increase IEC- phase interaction With the process of cell percentages (relative to the percentage in sample before enrichment procedures).Purifying is an example of enrichment.? IEC- phase interaction in certain embodiments, relative to the sample before enrichment procedures, in enriched sample as cell percentages With number increase at least 25-, 50- of cell (such as APC (for example, dendritic cells) or tumour cell), 75-, 100-, 150-, 200-, 250-, 300-, 350- times suitably increase 100-200 times.In specific embodiments, thin in IEC- interaction The antibody of born of the same parents upper surface marker can be attached on solid support to allow to separate.Separating step may include using antibody Magnetic bead is (for example, Miltenyi^TMPearl) Magnetic Isolation, affinity chromatography, using be attached to solid matrix antibody carry out " elutriation ", Or any other convenient technology (such as detection wind lidar).In specific embodiments, IEC- interaction cell It is dendritic cells, suitably its use is specific to CD11c antibody and is enriched with, and this antibody and magnetic bead are conjugated, and magnetic thin Born of the same parents' separator is for separating CD11c⁺Cell.There is provided the other technologies especially accurately separated includes FACS.Cell is once heavy On slide, they can be fixed product, and be detected with the antigen binding molecules (such as labelled antibody) of label, with cell spy Specific fashion detects Th1 immune state biomarker.

It is specific to the antibody of Th1 immune state biomarker, such as anti-PD-L2 antibody, can directly be conjugated to fluorescence Marker, including fluorescein, FITC, rhodamine, texas Red, Cy3, Cy5, Cy7 and other fluorescent markers, and be equipped with The fluorescence microscopy of appropriate filter is under the microscope.Antibody can also be conjugated with enzyme, start to react after substrate appropriate is added, examine It measures and provides colored precipitate on the cell of PD-L2 albumen.It may then pass through standard light microscope observation slide.Alternatively, PD- The specific primary antibody of L2 can further with two anti-bindings that are conjugated with detectable part.Cell surface table can thus be assessed It reaches, and cell permeabilization solution (such as Triton-X and saponin(e) can be added to promote intracytoplasmic reagent infiltration (" Cell Biology:A Laboratory Handbook ", rolls up 1-111Cellis, and JE compiles (1994)；"Current Protocols in Immunology " rolls up I-III Coligan JE and compiles (1994)；Stites et al. (eds.) " Basic and Clinical Immunology " (the 8th edition), Appleton&Lange, Norwalk, Conn. (1994)；Mishell and Shiigi (eds.) “Selected Methods in Cellular Immunology”,W.H.Freeman and Co.,New York(1980)。

Immunohistochemistry and immunofluorescence or immunocytochemistry are closely similar, however tissue specimen PD- in principle L2 antibody is detected, such as different from cell suspending liquid.Fixed biopsy sample is simultaneously processed, and paraffin is optionally embedded in In, it is sliced if necessary, the cell or tissue slice then detected with heparitinase specific antibody is provided.Alternatively, cold Freezing tissue can be sliced on cryostat, then carry out antibody detection, avoid the fixed antigen masking induced.It is such as immunized glimmering Antibody in light or immunocytochemistry and detectable part (fluorescence or enzyme connect) coupling, and by glimmering for being immunized Method described in light then passes through fluorescence or Laser Scanning Confocal Microscope according to detection method used for detecting histotomy Observation.If using the detectable part of enzymatic after reaction product colour developing, can be observed by standard light microscope anti- Answer visualization (" Cell Biology:A Laboratory Handbook ", volume 1-111Cellis, the JE volume of product sediment (1994)；" Current Protocols in Immunology " rolls up I-III Coligan JE and compiles (1994)；Stites et al. (eds.) " Basic and Clinical Immunology " (the 8th edition), Appleton&Lange, Norwalk, Conn. (1994)；Mishell and Shiigi (eds.) " Selected Methods in Cellular Immunology ", W.H.Freeman and Co.,New York(1980))。

In specific embodiments, facs analysis be used for assess Th1 immune state biomarker expression (for example, The expression of PD-L2 and optional PD-L1).U.S. Patent number 4,172,227；4,347,935；4,661,913；4,667,830； 5,093,234；The general description of FACS device and method is provided in 5,094,940 and 5,144,224.Introduce cells into FACS machine It in device and is pipelined in the pond FACS, they are as individual cells by wherein.Laser beam is directed toward the pond FACS, passes through light To laser light scattering before electric diode collection, lateral laser light scattering passes through lens and is directed toward PMT pipe, is directed toward PMT1.Specific filter Other PMT pipes will be directed directly to carry out multi-variables analysis from sidewise scattered fluorescence.Lateral laser light scattering is that cell is big Small and granularity reflection, and can be used for identifying the cell mass in mixing sample.It can be managed by laser excitation and by PMT It collects to detect the cell with the anti-PD-L2 antibody label of fluorescence, can be by size and grain qualification cell type, or lead to It crosses and mixes additional cell surface marker object and identified, such as is disclosed above.In general, facs analysis is for determining specific egg White cell surface expresses (for example, expression of PD-L2 and optional PD-L1), therefore specific antibody can be used for detecting antigen and be in (for example, the expression of PD-L2 and optional PD-L1 on surface of dendritic cells) cell surface biomarkers are expressed in delivery cell group Detection.The specific antigen presentation cell subsets of surface PD-L2 albumen and optional PD-L1 are expressed (for example, CD11c⁺Dendron is thin Born of the same parents) it can be determined by size and grain size characteristic, or by the total dyeing with other cell surface marker object albumen come really It is fixed.

In specific embodiments, using permission while detection and/or the albumen capture array of quantitative a large amount of albumen.Example Such as, the low-density protein arrays on filter membrane, such as universal protein array system (Ge, 2000Nucleic Acids Res.28 (2): e3) allow the antigen using standard ELISA technology and scanning charge-coupled device (CCD) detector imaging arrangement.Also open Immuno-sensor arrays have been sent out, clinical analysis object can be detected simultaneously.Protein arrays can be used to detect in body fluid now Protein expression profiles, such as health or deceased subject and before drug therapy and treatment after subject serum in.

Exemplary Proteins capture array includes array (the commonly known as antibody of the antigen binding molecules comprising space addressing Array), it can promote the extensive parallel analysis for defining many albumen of protein groups or sub- protein groups.Antibody array has been demonstrated have Have specificity and acceptable background required characteristic, and it is some be obtained commercially (for example, BD Biosciences, Clontech, BioRad and Sigma).The a variety of methods for being used to prepare antibody array be reported (see, e.g., Lopez etc., 2003J.Chromatogr.B787:19-27；Cahill, 2000Trends in Biotechnology 7:47-51；The U.S. is special Sharp Shen Qing Publication 2002/0055186；U.S. Patent Application Publication 2003/0003599；PCT Publication WO03/062444；PCT is public Cloth WO03/077851；PCT Publication WO02/59601；PCT Publication WO 02/39120；PCT Publication WO01/79849；PCT Publication WO99/39210).The antigen binding molecules of array can recognize at least one son by cell or the albumen of cell mass expression in this way Collection, illustrated examples include growth factor receptors, hormone receptor, neurotransmitter receptor, catecholamine receptor, amino acid derived Object receptor, cytokine receptor, the receptor of extracellular matrix, antibody, agglutinin, cell factor, serpin, Protease, kinases, phosphatase, ras sample GTP enzyme, hydrolase, steroid hormone receptor, transcription factor, Features of The Heat Shock Transcription Factor, DNA binding protein, zinc finger protein, leucine zipper protein, homeodomain albumen, intracellular signal transduction regulatory factor and effect Answer the factor, Apoptosis-Related Factors, DNA composition-factor, DNA reparative factor, DNA recombinant factor and cell surface antigen.

Individually spatially different albumen capturing agent is usually attached to the supporter surface of usually plane or waveform.Often The physical support object seen includes slide, silicon, micropore, nitrocellulose or pvdf membrane and magnetic bead and other microballons.

As long as their coding is in order to which for identifying, the particle in suspension also is used as the basis of array；System includes The color coding (for example, being purchased from Luminex, Bio-Rad and Nanomics Biosystems) and semiconductor nano of microballoon Body is (for example, be purchased from the QDots of Quantum Dots^TM) and the bar code of pearl (be purchased from Smartbeads's UltraPlex^TM) and more metal micro stick (Nanobarcodes purchased from Surromed^TMParticle).Pearl can also be assembled into The planar array (for example, being purchased from LEAPS technology and BioArray Solutions) of semiconductor core on piece.Work as use When particle, individual albumen capturing agent is usually attached to individual particle, to provide space restriction or the interval of array.Particle Then it can be measured parallel in the hole of such as microtiter plate or in individual test tube individually but in a manner of compartmentation.

In operation, by optionally fragmentation to form the protein sample of peptide fragment (see, e.g., U.S. Patent application It announces 2002/0055186), is delivered to albumen capture array, and the array quilt under conditions of being suitable for albumen or peptide combines It washs to remove unbonded or non-specific binding the component of sample from the array.Then, using suitable detection system Come detect each feature for being bound to array albumen or peptide presence or amount.The amount for being bound to the albumen of the feature of array can phase Amount for being bound to the second albumen of the second feature of array determines.In certain embodiments, the second albumen in sample Amount be known or be known to be constant.

In specific embodiments, Th1 immune state biomarker is target polypeptide, uses at least one and target The antigen binding molecules of the immune interaction of polypeptide measure its level.In these embodiments, by the target polypeptide water of measurement The flat level for being normalized to reference polypeptide.Suitably, antigen binding molecules are fixed on solid or semisolid support.This In the illustrated examples of type, antigen binding molecules form a part of antigen binding molecules space array.In some embodiment party In case, pass through the level of antigen binding molecules of immunoassay (for example, using ELISA) measurement in conjunction with target polypeptide.

Proof is not present in sample or there are biomarker activity, it is raw to be to discriminate between expression specificity Th1 immune state The IEC- interaction cell mass of substance markers object and the not cell mass of expression specificity Th1 immune state biomarker are in addition Method.

The aggregation of 8.4 evaluation PD-L2 biomarkers

The present inventor further define aggregation of the PD-L2 on the cell surface of IEC- interaction cell indicate it is normal or Raised Th1 immune response.Therefore, in some embodiments, the biomarker value of Th1 immune state biomarker refers to Show the level or abundance of PD-L2, such as interacting by analysis IEC-, (such as APC (for example, dendritic cells) or tumour are thin for cell Born of the same parents) cell surface on PD-L2 aggregation determined by.

There are many cell surfaces that widely available measuring method is used to detect antigen presenting cell (for example, dendritic cells) PD-L2 aggregation.For example, PD-L2 ligand and/or PD-L2- specific antibody can be marked, and detect these labels with Show the aggregation of PD-L2.In an example of such measurement, make dendritic cells or tumour cell comprising PD-L2 It is contacted with the secondary antibody of PD-L2- specific antibody and the fluorescent marker in conjunction with PD-L2- specific antibody.Use confocal scanning Laser microscope can detecte the fluorescence issued from secondary antibody, to identify position (Van Steensel et al., 1995.J of PD-L2 Cell Sci 108:3003-3011).In another example, the cell and cell surface PD- on cell surface comprising PD-L2 The tagged ligand of L2 contacts, and analyzes cell surface PD-L2 aggregation, such as Kaufmann et al. by super-resolution microscope (2011.J Microsc.242 (1): 46-54), Huber et al. (2011.PLoS One.7 (9): e44776), Wang et al. (2014.Biochim Biophys Acta.1838 (4): 1191-1198) and Sams et al. (2014.J Biomed Opt.19 (1): 011021) described.

Alternatively, analyzing cell surface PD-L2 aggregation, such as Bellucci et al. by proximity assay in situ (2014.Methods Mol Biol.1174:397-405), Barros et al. (2014.Breast Cancer Res Treat.144 (2): 273-85) and Pacchiana et al. (2014.Histochem Cell Biol.142 (5): 593-60) institute Description.

In other embodiments, FRET and FRAP microscopy can be used for analyzing PD-L2 aggregation, such as Wallrabe et al. (2003.Biophys J.85 (1): 559-571), Wallrabe et al. (2003.J Biomed Opt.8 (3): 339-346) and De Heus et al. (2013.Methods Cell Biol.117:305-321) is described.

The other methods for analyzing PD-L2 aggregation include: image correlation spectrometry, such as Petersen et al. (1998.Faraday Discuss. (111): 289-305), Kozer et al. (2013.Mol Biosyst.9 (7): 1849- 1863) and Ciccotosto et al. (2013.Biophys J.104 (5): 1056-1064) described；Analysis of electric field, such as Giugni et al. (1987.J Cell Biol.104 (5): 1291-1297) and Zhang et al. (2011.PLoS One.6 (10): E26805) described；Electron microscope, such as Plowman et al. (2005.Proc Natl Acad Sci USA.102 (43): 15500-15505) and D'Amico et al. (2008.Micron.39 (1): 1-6) is described；Electronics freezes tomography, such as Gold et al. (2014.Nat Commun.5:4129) is described；Nano particle (NP) is immune labeled with plasmon coupling microscope (PCM) combination, such as Wang et al. (2012.Nano Lett.12 (6): 3231-3237) and Rong et al. (2012.PLoS One.7 (3): e34175) described；Radioactive source activation (EMARS) analysis that enzyme mediates, such as Miyagawa-Yamaguchi et al. (2014.PLoS One.9 (3): e93054) and Kotani et al. (2008.Proc Natl Acad Sci USA.105 (21): It is 7405-7409) described；With quantum point analysis, such as Li et al. people is (2010.Biophys J.98 (11): 2554-2563) described.

Known many ligands with PD-L2 specificity, can be used for analysis of agglomeration.Many in these also is used as root According to therapeutic agent of the invention.For example, it is contemplated that the suitable antibodies of any combination PD-L2 polypeptide can be used for implementing the present invention.With above-listed The non-limiting example of such antibody is gone out.

In specific embodiments, antibody includes the area Fc of immunoglobulin.Alternatively, or in addition, antibody is multivalence (for example, divalent) antibody.

Any PD-L2 ligand is suitable for the invention these embodiments, the ligand including following classification: albumen small has Machine molecule, carbohydrate (including polysaccharide), polynucleotides, lipid etc..The representative example of such ligand include PD-1 polypeptide, - 9 polypeptide of galactose agglutinin and repellency instruct molecule b (RGMb).

8.5 assessment Th1 immune state biomarker nucleic acid

In some embodiments, by determine IEC- interact cell (such as APC (such as dendritic cells) or tumour it is thin Born of the same parents) in biomarker transcribed nucleic acid object level come monitor biomarker expression.It can be by many standard techniques from life In object sample extract RNA (referring to Current Protocols in Molecular Biology " roll up I-III Ausubel, R.M compiles (1994)；Ausubel et al. " Current Protocols in Molecular Biology ", John Wiley and Sons,Baltimore,Md.(1989)).Cell lysing methods based on guanidine are able to carry out RNA separation, are followed by chlorination Then caesium step gradient carries out RNA precipitate and resuspension for separating RNA from other cellular macromolecules, this is not earlier not Too common RNA separation method (Glisin, Ve. et al. (1973) Biochemistry 13:2633).Alternatively, can be with one-step method Separation RNA (U.S. Patent number 4,843,155 and Puissant, C and Houdebine L.M (1990) Biotechniques 8: 148-149).One-stage procedure includes carrying out RNA extraction using guanidinium isothiocyanate, then carries out phenol/chloroform/isoamyl alcohol extracting It takes, promotes the separation of total serum IgE and other cell proteins and DNA.Commercially available single dip formulation based on the above principles can be used, including Such as use TRIZOL reagent (Life Technologies, Gaithersburg, Md).

Th1 immune state biomarker RNA/ gene expression can be monitored by some other standard techniques, explanation Property example includes Northern trace and dot blot assay, primer extend, RNA enzyme protection, RT-PCR, in situ hybridization and core Piece hybridization.

Specificity T h1 immune state biomarker RNA sequence can pass through label probe and trace RNA extracted as above Preparation is hybridized and is easily detected.In Northern engram analysis, Denaturing Agarose Gel electricity is carried out to the RNA of classification Swimming, this prevents RNA appearance from may inhibit the isolated secondary structure based on size.Then RNA is shifted by capillary transfer Onto nylon or nitrocellulose filter support, and the labeled oligonucleotide probe complementary with biomarker sequence can be used Detected (Alwine, et al. (1977) Proc.Natl.Acad.Sci.USA 74:5350-5354 and Current Protocols in Molecular Biology " rolls up I-III Ausubel, and R.M. compiles (1994)；Ausubel et al. “Current Protocols in Molecular Biology”,John Wiley and Sons,Baltimore,Md. (1989))。

Alternatively, unassorted RNA can be fixed on nylon or nitrocellulose filter, and pass through Slot/Dot trace point Similarly detection biomarker-is specific expressed for analysis.The Slot/Dot trace of RNA can be by preparing by hand or using Manifold (manifold) device is constructed, and compares hybridization signal (Chomczynski P. by densitometric scan promotion (1992) Anal.Biochem.201:134-139).

Primer extend is that another kind can complete the quantitative method of RNA.By using enzyme reverse transcriptase extension primer, primer It extends to and draws the end 5' of specific RNA and provide additional benefit.In this case, primer is and biomarker mRNA A part of complementary oligonucleotides (or restriction fragment).Prime end is marked, and allows and template biological marker MRNA hybridization.After hybridization, it is incorporated into and template life by the way that reverse transcriptase extension primer is added, and by unlabelled deoxynucleotide In the single stranded DNA of substance markers object mRNA complementation.Then DNA is analyzed on sequencing gel, the length of extension primer is for drawing The position 5' of mRNA, and extension products yield reflection sample in RNA abundance (Jones et al., (1985) Cell 42: 559-572 and Mierendorf R.C and Pfeffer, D. (1987) Methods Enzymol.152:563-566).

RNA enzyme protection measuring method provides the method for super-sensitive quantitative biomarker object RNA, even if in low abundance It is also such.In protection measurement, ribonucleotide that there is high specific activity, complementary with biomarker RNA is generated The sequence specific hybridization of probe, and hybridize with sample RNA.Then free to remove with ribonucleic acid enzymatic treatment hybridization reaction object Probe hybridizes the complete segment of annealed probe with the homologous organisms flag sequence in sample RNA.Then by sequencing gel Upper electrophoresis analytic plate section, the at this time appropriately sized probe fragment of observable (Zinn K. et al. (1983) Cell 34:865-879 With Melton S.A., et al. (1984) .Nucl.Acids Res.12:7035-7056).

RT-PCR is the another way that can analyze biomarker expression.RT-PCR use and biomarker mRNA Complementary deoxynucleotide primer from RNA sample by preparing cDNA using reverse transcriptase.Once generating cDNA, pass through addition Deoxynucleotide and the archaeal dna polymerase to work at high temperature, pass through PCR amplification cDNA.Pass through primer annealing Repetitive cycling, incorporation deoxynucleotide promote cDNA to extend, then carry out chain denaturation, and the amplification of sequence needed for occurring, generation can The appropriately sized segment detected by agarose gel electrophoresis.Best reverse transcription, hybridization and amplification condition will draw according to used Experimental method that the sequence of object and target composition and length and operator select and change.Various guidances can be used to select Suitable primer sequence and hybridization conditions (see, e.g. Sambrook et al. 1989, Molecular Cloning, Laboratory Manual, (volume 1-3) Cold Spring Harbor Press, N.Y.；And Ausubel et al. 1989, Current Protocols in Molecular Biology,Green Publishing Associates and Wiley Interscience,N.Y.)。

In situ hybridization offer can be used for detecting and positioning cell/tissue specific biomarkers rna expression.What is marked is anti- Adopted rna probe hybridizes (In with the mRNA for being fixed on microslide in individual cell or in the histotomy of processing Situ Hybridization:Medical Applications (eds.G.R.Coulton and J.de Belleroche) Kluwer Academic Publishers,Boston(1992)；In Situ Hybridization:In Neurobiology；Advances in Methodology (eds.J.H.Eberwine, K.L.Valentino and J.D.Barchas) Oxford University Press Inc., Britain (1994)；And In Situ Hybridization:A Practical Approach (ed.D.G.Wilkinson) Oxford University Press Inc., Britain (1992)).Many heterotope systems are developed to observe the DNA probe of label, comprising: a) based on glimmering The direct detecting method of light, b) it is combined using the DNA probe of digoxin and biotin labeling with fluorescence detection method and c) is made It is combined with the DNA probe of digoxin and biotin labeling with antibody-enzyme assay method.When the antisense RNA probes of fluorescent marker When hybridizing with cell RNA, the probe that fluorescence microscope directly observes hybridization can be used.The direct fluorescent dye mark of nucleic acid probe Note eliminates the needs (for example, system based on antibody) to multilayer detection program, allows quickly to handle and also reduce non- Specific background signal, therefore general and super-sensitive means are provided to identify biomarker gene expression.

For chip hybridization using the biomarker specific oligonucleotide that is attached on solid matrix, solid matrix can be by Particulate solid composition, such as it is designed as nylon filter, slide or silicon wafer (Schena et al. (1995) of microarray Science 270:467-470).Microarray is known in the art and by surface composition, is produced on such surface with gene The corresponding probe of object (such as cDNA) sequence can hybridize or be incorporated in known location specifically to detect biomarker base Because of expression.

Quantifying for hybridization complex is well known in the art, and can be by any one of several method come real It is existing.These methods are typically based on the detection of label or marker, for example, this field standard use any radioactivity, fluorescence, life Object or enzyme label or tag.Label can be applied to oligonucleotide probe or the RNA from biological sample.

In general, suitably realizing that mRNA is quantitative together with calibration curve, so as to accurately determine mRNA.In addition, fixed Amount is originated from the transcript of biological sample preferably by realizing compared with normal specimens, and this sample is characterized in that being checked Transcript expression pattern it is normal.

8.6 derive biomarker value

Biomarker value can be the biomarker value of measurement, be for subject's biomarker measured directly The value of object, or can be " derivation " biomarker value, it is derived from the biomarker value of one or more measurements Value, such as the biomarker value by the way that function to be applied to one or more measurements.As it is used herein, having applied function Biomarker be referred to as " biomarker of derivation ".

Biomarker value can be determined with any one of various ways well known in the art.For example, biology mark The comprehensive description that note object value determines can be found in International Patent Publication No. WO 2015/117204, and entire contents are by drawing With being incorporated herein.In one example, the method for determining biomarker value may include measurement biomarker value, such as logical It crosses and subject or the sample obtained from subject is tested.

More typically, however, the step of determining biomarker value includes receiving electronic processing device or with its other party Formula obtains the biomarker value for previously having measured or having derived.This may include for example depositing from the data of such as remote data base Biomarker value is retrieved in storage, obtains the biomarker value being manually entered using input equipment etc..Suitably, it indicates The combination of multiple biomarker values can be used to determine in object, and indicant at least partly indicates Th1 immune state.Assuming that making This method is executed with electronic processing device, optionally shows or otherwise provide a user the instruction of indicant.

In some embodiments, combine biomarker value, such as by adding, multiplied by, subtract or divided by biology mark Object value is remembered to determine indicant value.Executing this step allows multiple biomarker values to be combined into single indicant value, More useful and simple mechanism is provided to allow indicant to be explained, and is accordingly used in determining the Th1 immune state of subject.

It should be appreciated that within a context, the biomarker used in the above-mentioned methods can limit Th1 immune state Biomarker spectrum comprising the biomarker (for example, at least one biomarker) of minimal number, while keeping enough Performance make biomarker spectrum can be used for clinically relevant determination.The number of biomarker used is reduced to the maximum extent It can reduce to the maximum extent and carry out diagnosis or the relevant cost of prognosis test, and the polypeptide biomarker the case where Under, allow to be tested using relatively simple technology, such as the cell sorting (FACS) and immunohistochemistry of fluorescent activation, And allow quickly to be tested in clinical setting.In this respect, instruction can be by the instruction that method described herein provides The figure or alphanumeric representation of object value.Alternatively, however, this instruction can be indicant value and predetermined threshold or range Comparison result, or can be the instruction of Th1 immune state.

Allow clinician or other medical operators that test result is easily explained in addition, generating single indicant value, So that the reliable diagnosis that test can be used in clinical setting.

Only as explanation, the method for suitably determining indicant includes determining at least one biomarker value, wherein giving birth to Substance markers object value is value measure at least one Th1 immune state biomarker of subject or derivation, and extremely Partially instruction is derived from the concentration or abundance of Th1 immune state biomarker in the sample of subject, and wherein at least One Th1 immune state biomarker include IEC- interaction cell (including APC (for example, dendritic cells) and tumour it is thin Born of the same parents) PD-L2.Suitably, Th1 immune state biomarker spectrum also comprising IEC- interaction cell (such as APC (for example, Dendritic cells) or tumour cell) PD-L1 as Th1 immune state biomarker.Biomarker value is commonly used in determination Indicant, for determining the Th1 immune state of subject.In some embodiments, indicant is that a pair of of Th1 immune state is raw The instruction of substance markers object (for example, PD-L2 and PD-L1) concentration rate.Therefore, if biomarker value indicates that shape is immunized in Th1 The concentration of state biomarker, then the biomarker value derived usually (although the not absolutely) ratio based on biomarker value Rate.

Then, as generally known in the art and as described in more detail below, by using the biomarker of derivation The biomarker value of derivation (such as is compared by object value as indicant value or by executing additional treatments with reference value Deng), indicant is determined using the biomarker value of derivation.

The biomarker value of derivation, such as additive model are combined using composite function；Linear model；Supporting vector Machine；Neural network model；Random Forest model；Regression model；Genetic algorithm；Annealing algorithm；Weighted sum；Neighborhood Model；With it is general Rate model.In some embodiments, biomarker value is to be directed to PD-L2 and PD-L1 measurement or derivation, and pass through Biomarker value is combined to determine indicant.In some embodiments, indicant and indicant reference are compared, It is middle that Th1 immune state is determined according to comparison result.Indicant reference can be derived from the finger that individual amount determines in reference group Show object.Reference group generally includes the individual with different characteristic, such as different sexes and/or multiple individuals of race, is based on The different group of different characterizing definitions refers to the indicant of subject and the indicant from the individual with similar features It is compared.Reference group can also include multiple healthy individuals, it is known have enhancing Th1 immune state it is multiple individual, It is known that there are multiple individuals that are reducing or lacking Th1 immune state, show cancer (suitably, metastatic cancer) clinical symptoms It is multiple individual or display pathogenic infection (for example, malaria) clinical symptoms multiple individuals.

In specific embodiments, using at least one electronic processing device (such as suitably programmed department of computer science System etc.) come the method that executes determination indicant of the invention.In this case, by being received from measurement or other proportioning devices This data or by being retrieved from database etc., electronic processing device usually obtain biology of at least one measurement Marker value.Then, processing unit determines indicant by any suitable means, such as is exempted from by calculating the first Th1 of instruction The value of the ratio of epidemic disease state biomarker and the 2nd Th1 immune state biomarker concentration.

Then, the expression of indicant, such as symbol or alphanumeric by generating indicant can be generated in processing unit It indicates, the alphabetical number of figure instruction or subject Th1 immune state of the indicant with one or more indicants with reference to compared with Word instruction.

The method of determination indicant of the invention generally includes to obtain sample from subject, and the subject usually has At least one clinical indication of Th1 related disease (for example, pathogenic infection or cancer), wherein the sample includes a kind of or more Kind Th1 immune state biomarker (for example, PD-L2 and optional PD-L1), and quantitatively or otherwise assess in sample extremely Few one kind (for example, 1,2,3,4,5,6,7,8,9,10 or more) Th1 immune state biomarker, to determine biomarker Value.Any suitable technology can be used to realize in this, and depends on the property of Th1 immune state biomarker.Suitably Ground, a bulk measurement or derivation Th1 immune state biomarker value correspond to the water of each Th1 immune state biomarker Flat, abundance or amount, or corresponding to the function for being applied to this level or amount.For example, present invention determine that indicant method In some embodiments, if using the indicant of multiple Th1 immune state biomarkers based on the ratio of peptide concentration, This process is generally included through any method known in the art (including immunofluorescence or pass through functional examination) come quantitative more Peptide.

In some embodiments, it is established by one or more Th1 immune state biomarker values of determination tested The Th1 immune state of person, wherein individual Th1 immune state biomarker value indicates the sample in subject or obtained from subject The value of Th1 immune state biomarker measured in product or derivation.Referred to as " shape is immunized in sample Th1 to these biomarkers State biomarker ".According to the present invention, sample Th1 immune state biomarker, which corresponds to, refers to Th1 immune state biology mark Remember object (herein also referred to as " corresponding Th1 immune state biomarker ")." corresponding Th1 immune state biomarker " refers to Th1 immune state biomarker in structure and/or is functionally similar to reference to Th1 immune state biomarker, such as Shown in SEQ ID NO:1 (PD-L2) and SEQ ID NO:56 (PD-L1).Representative corresponding Th1 immune state biomarker Allelic variant (identical locus), homologue (different genes including reference Th1 immune response biomarker gene Seat) and ortholog (different biological) expression product.With reference to the Th1 of Th1 immune state biomarker gene and coding The Nucleic acid variant of immune state biomarker polypeptide can contain nucleotide substitution, missing, transversion and/or insertion.Variation can be with Occur in any of code area and noncoding region or both.These variations can produce conservative and nonconserved amino acid and take Generation (compared with coded product).For nucleotide sequence, due to the degeneracy of genetic code, conservative variant includes coded reference Those of the amino acid sequence of Th1 immune state polypeptide sequence.

Th1 immune state biomarker includes and the amino with reference to Th1 immune state biomarker polypeptide accordingly Acid sequence shows the amino acid sequence of substantially sequence similarity or identity.In general, corresponding to reference amino acid sequence Amino acid sequence, with selected from SEQ ID NO:1 to any of 9 reference amino acid sequence will show at least about 50,51,52, 53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、 78,79,80,81,82,83,84,85,86,97,88,89,90,91,92,93,94,95,96,97,98 or 99% sequence is similar Property or identity.

In some embodiments, the calculating of the sequence similarity or sequence identity between sequence carries out as follows:

In order to determine the percentage identity of two amino acid sequences or two nucleic acid sequences, for the purpose most preferably compared Sequence is compared (for example, can introduce vacancy in one or two of the first and second amino acid or nucleic acid sequence So as to optimal comparison, nonhomologous sequence can be ignored in order to omparison purpose).In some embodiments, compare to be omparison purpose Pair reference sequences length be reference sequences length at least 30%, typically at least 40%, more generally at least 50%, 60%, Even more typically at least 70%, 80%, 90%, 100%.Then the amino acid of more corresponding amino acid position or nucleotide position Residue or nucleotide.When the position in First ray is accounted for by the same amino acid residue of corresponding position in the second sequence or nucleotide According to when, then molecule is identical in the position.Amino acid sequence is compared, when the position in First ray is by the second sequence When the same or similar amino acid residue (i.e. conservative substitution) of corresponding position occupies, then molecule is similar in the position.

Percentage identity between two sequences is the same amino acid residue number that sequence is shared on each position Function considers the number in vacancy and the length in each vacancy, needs to introduce vacancy to obtain the optimal comparison of two sequences.Phase Than under, the percent similarity between two sequences is the identical and similar amino acid residue number that sequence is shared on each position Purpose function considers the number in vacancy and the length in each vacancy, needs to introduce vacancy to obtain the best ratio of two sequences It is right.

The percentage identity or percent similarity between the comparison and sequence of mathematical algorithm completion sequence can be used Determination.In certain embodiments, using 62 array of Blossum or PAM250 array, 16,14,12,10,8,6 or 4 sky Position weight and 1,2,3,4,5 or 6 Length Weight, using Needleman and W ü nsch (1970, J.Mol.Biol.48: 444-453) algorithm determines that percentage identity or similitude between amino acid sequence, the algorithm have been included into GCG software GAP program (can be obtained from http://www.gcg.com) in packet.In specific embodiments, it uses NWSgapdna.CMP array, 40,50,60,70 or 80 gap weight and 1,2,3,4,5 or 6 Length Weight, use GAP program (can obtain from http://www.gcg.com) in GCG software package determines the identity hundred between nucleotide sequence Divide ratio.Non-limiting parameter group (unless otherwise stated, the parameter that should be used) includes the gap penalty with 12,4 sky Position extend point penalty and 5 frameshift gap point penalty Blossum 62 score array.

In some embodiments, the percentage identity between amino acid or nucleotide sequence or similitude can be used The algorithm of E.Meyers and W.Miller determines (1989, Cabios, 4:11-17), has been incorporated into (version in ALIGN program 2.0), using PAM120 weight residue table, GAP LENGTH PENALTY 12, gap penalty 4.

Nucleic acid and protein sequence described herein may be used as " search sequence " to scan for public database, such as To identify other family members or correlated series.Altschul et al. (1990, J Mol Biol., 215:403- can be used 10) NBLAST and XBLAST program (version 2 .0) carries out such search.Using NBLAST program, scoring=100, word length =12 carry out BLAST nucleotide search, to obtain the nucleotide sequence with 53010 nucleic acid molecule homologous of the invention.It can use XBLAST program, scoring=50, word length=3 carry out BLAST protein searches, to obtain the ammonia homologous with protein molecular of the invention Base acid sequence.Compared to obtain vacancy for comparative purposes, using vacancy BLAST, as Altschul et al. (1997, Nucleic Acids Res, 25:3389-3402) it is described.When using BLAST and gapped BLAST programs, it can be used corresponding The default parameters of program (for example, XBLAST and NBLAST).

Corresponding Th1 immune state biomarker polynucleotides further include exempting under following stringent conditions with reference Th1 Epidemic disease state biomarker polynucleotides or the nucleic acid sequence of its complement hybridization.As used herein, term " it is low strict, in Very hybridize Deng stringent, high stringency or under high stringency conditions " describe the condition of hybridization and washing." hybridization " used herein indicates The pairing of complementary nucleotide sequence is to generate DNA-DNA heterozygote or DNA-RNA heterozygote.Complementary base sequence is to pass through base Those of pairing rules correlation sequence.In DNA, A and T are matched, and C and G are matched.In RNA, U and A are matched, and C and G are matched.? This respect, the term as used herein " matching " and " mispairing " refer to the hybridization potentiality that nucleotide is matched in complementary nucleic acid chain.? The nucleotide matched effectively hybridizes, such as A-T the and G-C base-pair of above-mentioned classics.Mispairing is the nucleotide that cannot effectively hybridize Other combinations.

The guidance for carrying out hybridization reaction can be found in Ausubel et al. (1998, ibid) 6.3.1-6.3.6 section. Aqueous and non-aqueous methods are described in this bibliography, and can be used any.Low stringency condition is mentioned above Including and include at least about 1%v/v at least about 15%v/v formamide and at least about 1M at least about 2M salt, at 42 DEG C Lower hybridization, and at least about 1M are used to wash at 42 DEG C at least about 2M salt.It is pure that low stringency condition may also include 1% ox blood Albumen (BSA), 1mM EDTA, 0.5M NaHPO₄(pH7.2), 7%SDS hybridizes at 65 DEG C, and (i) 2 × SSC, 0.1% SDS；Or (ii) 0.5%BSA, 1mM EDTA, 40mM NaHPO₄(pH 7.2), 5%SDS are washed at room temperature.Low stringency condition An embodiment be included at about 45 DEG C the hybridization in 6 × sodium chloride/sodium citrate (SSC), then 0.2 × SSC, (for low stringency condition, the temperature of washing can increase to 55 DEG C) is washed twice in 0.1%SDS at least 50 DEG C.It is medium tight Glazing bar part includes and includes at least about 16%v/v at least about 30%v/v formamide and at least about 0.5M at least about 0.9M salt, For hybridizing at 42 DEG C, and at least about 0.1M is washed at 55 DEG C at least about 0.2M salt.Medium stringency condition can also wrap Include 1% bovine serum albumin(BSA) (BSA), 1mM EDTA, 0.5M NaHPO₄(pH7.2), 7%SDS is used to hybridize at 65 DEG C, and (i) 2 × SSC, 0.1%SDS；Or (ii) 0.5%BSA, 1mM EDTA, 40mM NaHPO₄(pH 7.2), 5%SDS are used for It is washed at 60-65 DEG C.One embodiment of medium stringency condition is included at about 45 DEG C and hybridizes in 6 × SSC, then 60 One or many washings are carried out at DEG C in 0.2 × SSC, 0.1%SDS.High stringency conditions include and include at least about 31%v/v It is used to hybridize at 42 DEG C at least about 50%v/v formamide and about 0.01M to about 0.15M salt, and about 0.01M to about 0.02M Salt at 55 DEG C for washing.High stringency conditions may also comprise 1%BSA, 1mM EDTA, 0.5M NaHPO₄(pH 7.2), 7% SDS is for 65 DEG C of hybridization, and (i) 0.2 × SSC, 0.1%SDS；Or (ii) 0.5%BSA, 1mM EDTA, 40mM NaHPO₄(pH 7.2), 1%SDS, for more than 65 DEG C at a temperature of wash.One embodiment of high stringency conditions is included at about 45 DEG C Hybridize in 6 × SSC, then carries out one or many washings at 65 DEG C in 0.2 × SSC, 0.1%SDS.

In certain embodiments, corresponding Th1 immune state biomarker polynucleotides are in very high stringency item The polynucleotides hybridized under part with disclosed nucleotide sequence (for example, SEQ ID NO:3 or SEQ ID NO:4).Very Gao Yan One embodiment of glazing bar part is included in 0.5M sodium phosphate, hybridizes in 7%SDS at 65 DEG C, then in 0.2 × SSC, 1%SDS It washed once at 65 DEG C or repeatedly.

Other stringent conditions are well known in the art, and technical staff will appreciate that manipulation various factors to optimize The specificity of hybridization.Optimizing the stringency finally washed can be used for ensuring that height hybridizes.Related detailed example, referring to Ausubel et al., see above 2.10.1 to 2.10.16 pages and Sambrook et al. (1989, ibid) the 1.101st to 1.104 section.

9. kit

All required reagents needed for detection and quantitative Th1 immune state biomarker of the invention can be in kit In fit together.In some embodiments, kit includes to allow quantitative at least one Th1 immune state biomarker Reagent.In some embodiments, the kit includes: (i) allows quantitative first Th1 immune state biomarker The reagent of (for example, determining abundance or level)；(ii) allows quantitative 2nd Th1 immune state biomarker (for example, determining Abundance or level) reagent.In some embodiments, kit also includes that (iii) optionally allows quantitative 3rd Th1 that shape is immunized The reagent of state biomarker (for example, determining abundance or level)；(iv) optionally allows quantitative 4th Th1 immune state biology The reagent of marker (for example, determining abundance or level).Suitably, Th1 immune state biomarker is PD-L2 and PD-L1 One or both of.

In the context of the present invention, " kit " is understood to mean that containing difference necessary to implementation the method for the present invention The product of reagent is packed to allow it to transport and store.Material suitable for packing this kit forms includes Crystal, plastics (polyethylene, polypropylene, polycarbonate etc.), bottle, bottle, paper, envelope etc..In addition, kit of the invention can With comprising for simultaneously, sequence or the specification that forms of difference contained by kit is used alone.Specification can be printing material Form, or be capable of direction memory book electronics support form, they are read by subject, such as electronics Storage medium (disk, tape etc.), optical medium (CD-ROM, DVD) etc..Alternatively, in addition, medium may include offer specification Internet address.

The reagent for allowing quantitative Th1 immune state biomarker includes allowing quantitative Th1 immune state biomarker Compound or material or compound or material group.In specific embodiments, compound, material or compound or material The group of material allows to determine the level or abundance of polypeptide (i.e. PD-L2 polypeptide).

Kit also optionally includes for detecting appropriate reagent, the positive and the negative control of label, washing solution, print Mark film, microtiter plate, dilution buffer etc..For example, the detection kit based on albumen may include that (i) Th1 immune state is raw Substance markers object polypeptide (for example, PD-L2 polypeptide and optional PD-L1 polypeptide, can be used as positive control), (ii) specific binding The antibody of Th1 immune state biomarker polypeptide.Alternatively, the detection kit based on nucleic acid may include (i) Th1 immune state Biomarker polynucleotides (for example, PD-L2 polynucleotides and optional PD-L1 polynucleotides, can be used as positive control), (ii) primer or probe that specificity hybridizes with Th1 immune state biomarker polynucleotides.It further include being suitable for expanding core The enzyme of acid, including various polymerases (reverse transcriptase, Taq, Sequenase^TM, DNA ligase etc., this depends on nucleic acid amplification used Technology), deoxynucleotide and buffer, to provide the necessary reaction mixture for amplification.Such kit usually will also be with Suitable mode includes the different vessels for every kind of independent reagent and enzyme and every kind of primer or probe.

In specific embodiments, kit further includes the above and broadly described immunological regulation of elsewhere herein Agent.

Kit can also have (for example, one or more) various devices and for carrying out one of measurement described herein (for example, one or more) reagent；And/or Th1 immune state biomarker gene expression is quantified using this kit Printing description.

Reagent described herein optionally in conjunction with detectable label can be rendered as microfluidic card, chip or room, The form of microarray or kit, be suitable for embodiment or hereafter described in measuring method be used together, such as it is described herein RT-PCR or Q round pcr.

10. diagnostic method

Indicant can also be used in determine subject with disease relevant to undesirable Th1 state of immune response can It can property.In this case, this is usually by the way that indicant and the reference of at least one indicant to be compared to realize, indicant With reference to indicate disease, and possibility is determined according to comparison result.It can be used for the Th1 correlation disease of diagnosing and treating according to the present invention The non-limiting example of disease include infectious diseases as described herein (especially virus infection) and proliferative diseases (for example, Metastatic cancer).

In such embodiment, the reference of at least one indicant is the indicant determined for reference group Distribution.For example, if subject occur pathogenic infection clinical symptoms (for example, hepatitis virus, fungal infection such as Aspergillus, Human immunodeficiency virus (HIV), malaria, typhoid fever, cholera, herpesviral, Chlamydia and HPV), then reference group is by with phase Same or similar disorder individual composition, and the indicant that will be used to compare subject.

In some embodiments, using the reference group of more than one individual, to determine subject with this disease A possibility that sick.For example, the first reference group is made of previous diagnosis and the known individual with disease of interest, the second reference Group is by being diagnosed as having the individual of health status to form.

In other embodiments, Th1 related disease is proliferative or hyperproliferative disorders, including it is any pernicious or Pre-malignant condition or due to proliferative capacity or any cell or tissue behavior of body in terms of functionality or other interference or abnormal Caused by derivative or relevant any disease.Therefore, method described herein can be used for diagnosing cancer, including assessment cancer A possibility that whether disease is metastatic cancer.

In order to should be readily appreciated that the present invention and try out, will be described by way of following non-limiting embodiment now Particularly preferred embodiment.

Embodiment

Embodiment 1

PD-L2 expression and people's severity of malaria on DC is negatively correlated

In order to determine whether PD-L1 and PD-L2 influences malaria immune, with 1800 plasmodium falciparums (P.falciparum) The red blood cell (pRBC) of infection infects the initial Healthy People volunteer of 7 malaria, and checks him within 7 days before attack and after attack Blood.In view of their important function (Wykes and Good, Nat Rev Microbial 6,864- in pathogenesis 867,2008), and because DC on PD-L1 and PD-L2 can lower T cell immune response (Brown et al., J.Immunol.170:1257-1266,2003；Freeman et al., J.Exp.Med.192:1027-1034,2000), the present invention People, which has studied, expresses the DC defined by CD11c.In all 7 volunteers, 90% DC expresses PD-L1 before infection, and There is no significant changes (Figure 1A) in the percentage of the 7th day DC for expressing the ligand of infection.On the contrary, although 80% DC is infecting It is preceding also to express PD-L2, but 5 in 7 individuals show the PD-L2 of significant decrease (17-57%) on the 7th day after infection⁺DC Percentage (Figure 1B).It is worth noting that, the 7th day after infection, parasitemia level expresses PD-L1 with PD-L2 on DC Percent ratio between observe significant negatively correlated (Fig. 1 C).In general, with have been generally acknowledged that PD-L2 as immunosupress The effect of agent in having the individual compared with low parasitemia, is observed associated on the contrary, after with falciparum infection Higher frequency expression PD-L2 DC.

Material and method

Human research

Carry out the method (McCarthy et al., PLoS One.6:e21914,2011) of clinical test (ClinicalTrials.gov identifier: NCT02389348) and PCR method (Rockett etc. for quantitative parasitemia People, Malar.J.10:48,2011) it has a detailed description elsewhere.Every participant provides informed consent.Eight health aspirations Seven (n=4 males in person；N=3 women) age be 19-55 years old (the median age 24 years old (quartile range, 21-37 Year)) research is participated in, to assess the validity of experimental anti-malarial therapy OZ439 and DSM265, agree to participate in being nested in facing respectively The sub- research in bed test.The research has obtained QIMR Berghofer Institute for Medical Research (QIMR) people's research ethics committee Approval.Volunteer receives about 1800 plasmodium falciparum pRBC in 2.0mL salt water by intravenous injection.(refer within 7th day Start to treat day calmly), participant is incorporated into research unit, and is collecting blood for applying research anti-malaria medicaments after studying Treatment.

Embodiment 2

PD-L2 expression and mouse malaria severity on DC is negatively correlated

In order to understand the biological associations of these data, next the present inventor has studied four kinds of malaria mouse models. They have selected four kinds of different Plasmodium species/strains to carry out infecting mouse, and every kind all shows unique biology and pathogenicity. When with 17XNL plants of non-lethality Plasmodium yoelii or Xia Shi Infected With Plasmodium WT mouse, the every 1-3 days parasitisms checked in blood Worm, infection is carried out with different rates, but two groups remove infection (Fig. 2A) in about 30 days.On the contrary, with Plasmodium yoelii YM The WT mouse of strain or Bai Shi mouse plasmodium ANKA infection shows serious but different lysis (Fig. 2 B；It is supervised according to table 3 and 4 It surveys).Compared with YM plants of Plasmodium yoelii infection, the parasitemia of Bai Shi mouse plasmodium is lower, because of Bai Shi mouse plasmodium sense The RBC of dye is lived in retirement from blood to deep tissue (including brain), leads to lethal encephalopathy.However, when clinical score >=4, institute (table 3 and 4) must be euthanized in 10 days by having the mouse of YM plants of Plasmodium yoelii and Bai Shi mouse Infected With Plasmodium.

The surface expression that PD-L1 and PD-L2 is checked on the DC from spleen, it is that helminth kills in mouse that it, which has been displayed, With adjust helminth specific immune response main portions (Yadava et al., Proc.Natl.Acad.Sci.U S A, 93: 4595-4599,1996).About 70% CD11c in the spleen of initial mouse⁺DC expresses PDL1, and this percentage exists Increase in Bai Shi mouse plasmodium and the mouse of Xia Shi Infected With Plasmodium, but during lethal or non-lethality Plasmodium Yoelii Infection Do not increase (Fig. 3 A).After all four malaria infections, compared with the DC from initial mouse, the DC for expressing PD-L1 is shown really Show that the surface expression levels (MFI) of PD-L1I increase, the mouse of non-lethality Xia Shi Infected With Plasmodium shows maximum increase (Fig. 3 B and 4A-F).On the contrary, the spleen DC from initial mouse < 5% expresses PD-L2.This people's blood DC with main expression PD-L2 is not Together, this separate sources for being likely to reflect they and blood and spleen.In addition, PD-L2⁺The percentage of DC is in all malaria senses Increase during dye, the significant bigger percentage (figure than in the mouse of lethal malaria is found in the mouse of non-lethality malaria 3C and 4A-E).Compared with the DC from initial mouse, with all helminth senses other than Bai Shi mouse plasmodium parasites In the mouse of dye, the MFI of the PD-L2 dyeing on PD-L2 expression DC also increases (Fig. 3 D and 4A-F).

Finally, the CD11c from lethal and non-lethality Plasmodium yoelii malaria⁺DC is in PD-L1 and PD-L2mRNA water Square face shows similar increase (Fig. 4 G), shows that the PD-L2 difference between these helminths noticed in Fig. 3 C depends on It is adjusted after transcription or albumen positions.It is worth noting that, the DC from lethal and non-lethality Plasmodium Yoelii Infection exists PD-L1 and PD-L2 expression and mRNA aspect surface having the same are horizontal, but in PD-L2⁺With non-PD-L1⁺The percentage of DC Middle difference.

Generally speaking, the result and hypothesis from all infection are consistent, i.e. PD-L2⁺The greater percentage of DC with it is advantageous Disease outcome is related.

Material and method

Mice study

The specific pathogen-free domestic C57BL/6J (wt) of 8-12 week old is obtained from Animal Resource Center (Perth, AUS) Female mice.Mouse is housed in QIMR zooscopy facility, and all programs are ratified by the QIMR animal welfare committee And monitoring.According to " Australia science purpose animal care and use working specification " (Australian National Health is ground with medicine Study carefully the committee), it is worked according to QIMR animal welfare approval number A0209-622M.Pass through Riken BRC, C57BL/6 background On PD-1 knock out (ko) (Pdcd1^-/-) mouse by doctor's T.Honjo friendship provide (Nishimura et al., Science.291: 319-322,2001).In these researchs using in C57BL/6J background PD-L2ko (Liang et al., Eur.J.Immunol.36:58-64,2006), PD-L1ko (Liang et al., Eur.J.Immunol.36:58-64,2006) and PD-1ko mouse confirms that gene is deleted by PCR test and/or flow cytometry.Using identical helminth, based on first The preceding research using similar measurement, to estimate sample size.

For the experiment with multiple groups, all mouse are infected first, are then assigned randomly in treatment group.Do not use Blind.

Parasitic infection and monitoring

With the 10 of the C57BL/6J mouse for being obtained from previous infection freshly⁵17XNL plants of Plasmodium yoelii, 10⁵Xia Shi plasmodium AS plants, 10⁴YM plants of Plasmodium yoelii or 10⁴ANKA plants of parasitic red blood cells (pRBC) of Bai Shi mouse plasmodium intravenously infect 3-6 The group of WT mouse.These helminth dosage had previously been shown generates apparent helminth blood within the about the same time Disease.Every 1-2 days preparation tail point blood films dye (Thermo Fisher using the Wright-Giemsa that Quick Dip is improved Scientific it) is dyed and is checked parasitemia, last up to 60 days.During parasitemia > 1%, pass through meter Number at least 300 RBC, and count in 20 visuals field of other times about 10,000 cell, to assess the percentage of pRBC.

The average percent parasitemia shown in several figures is the average percent of each total RBC of mouse in group pRBC.The physical symptom of the anaemia and disease of monitoring mouse daily, including posture (back of a bow), shortage activity and fur texture.Such as They show the significant distress sign as described in the following table 3 and 4 to fruit, then implement to be euthanized to mouse.

Table 3

Standard	0 grade	1 grade	2 grades
				Weight loss	< 10%	10 to 25%
Posture	Normally	Only in tranquillization hunchbacked (Hunching)	It is hunchbacked serious, influence activity
				Activity	Normally	Slightly reduced to moderate	It is stationary, unless being stimulated
Hair texture	Normally	Slightly to moderate fold	Serious fold/bad order
				Hemoglobin	Normally	<50g/L	<20g/L

The symptom that YM plants of Plasmodium yoelii includes anemicus respiratory distress, blood urine and complication (as gone into a coma and fainting from fear), but From being not suffering from cerebral malaria.During experiment, the distress of mouse is monitored by above-mentioned standard daily, is to determine to treat described in research It is no to cause mouse distress to the degree that be euthanized.By these evaluation criterias, if accumulation scoring reaches 3 or more, or If weight loss is more than 25%, poverty-stricken mouse is implemented to be euthanized.

Table 4

Symptom	Scoring	Metainfective number of days
			Hair fold	1	5
The back of a bow	1	5
			Gait is waved	1	6
Quadriplegia	1	6
			Twitch	1	6-7
Stupor	1	6-7

Bai Shi mouse plasmodium causes lethal encephalopathy, and the 7th day symptom is usually apparent after infection.Assessment is tired Long-pending, the mouse of accumulation scoring=4 is euthanized.It is worth noting that, the hair and the back of a bow of gauffer are general clinical symptoms, And (italic is shown) other symptoms are the symptoms of cerebral malaria.

Flow cytometry

The blood of processing or the single cell suspension of splenocyte are carried out with the antibody combination of fluorogen-conjugation as follows Label.Fixable Viability dyestuff eFluor780 (eBioscience) from analysis for excluding dead cell.Pass through stream Formula cell art tests being serially diluted for every kind of antibody in advance, to determine the optium concentration mainly measured.Anti- CD16/32 (clone 2.4G2, BD) for blocking non-specific Fc to combine.After fixed and permeabilization cell, with BD Pharmingen The cell inner mark object that Transcription Factor Buffer suit label asterisk indicates.Use BD LSR Fortessa Flow cytometer and BD FACSDiva software carry out data acquisition.Using FCS express (De Novo Software) or FlowJo (Tree Star) carries out data analysis.

Table 5

Embodiment 3

PD-L2 is necessary to survival and control helminth

In order to determine contribution of the PD-L2 to malaria control helminth, next the present inventor has checked compared with wt mouse, In PD-L2ko mouse Plasmodium yoelii 17XNL plants infection result (Liang et al., Eur.J.Immunol.36:58-64, 2006) (in C57BL/6J background).All wt mouse remove infection (Fig. 5 A) in 27 days.

However, PD-L2ko mouse has parasitemia significantly more higher than wt mouse, and institute after the 13rd day There are these dead mouses or is performed euthanasia (Fig. 5 A and Fig. 6 A) since clinical score >=4 (Fig. 6 B) was needed at the 19th day.Cause This, PD-L2 expression be helminth control and from 17XNL plant of Plasmodium yoelii infection in survival necessary to.

In order to confirm to observe, i.e. PD-L2 is necessary to surviving in 17XNL plants of Plasmodium yoelii infection, when discovery is posted When infested detectable in blood, next inventor uses monoclonal antibodies block PD-L2.For the experiment, wt mouse is used 17XNL plants of Plasmodium yoelii are infected and give within 4 days anti-PD-L2 or control rats IgG after infection, and every 3-4 days are applied With until 14-18 days after infection.All wt mouse for receiving rat IgG survive in 32 days and remove infection (Fig. 5 B and figure 6C).In contrast, although at anti-PD-L2 (Fig. 5 B and Fig. 6 C) similar with the controlling extent of helminth in control antibodies treatment group, But due to serious symptom, 100% infecting mouse for giving PD-L2 blocking antibody is dead or (figure was euthanized at the 19th day 6D).This is contrasted with PD-L2ko mouse, and PD-L2ko mouse shows significant higher parasitemia after the 13rd day (Fig. 5 A) shows that antibody does not completely inhibit function, or before blocking, 4 days PD-L2 funtion parts improve immune.

The effect in another non-lethality infection is being prevented in order to further explore PD-L2, is such as being directed to Plasmodium yoelii 17XNL plants of experiment treats (Fig. 5 C with non-lethality Xia Shi plasmodium malaria infection wt mouse and with anti-PD-L2 or rat IgG With Fig. 6 E).(the 8th day during acute infection；Pay attention to logarithmic scale), it survives from two groups of mouse, but the blocking of PD-L2 Parasitemia is significantly increased, causes the chronic infection phase parasitemia of (> 21 days) generally higher and helminth is clear Except (arrow indicates that helminth is removed in the mouse that rat IgG is handled for delay 4 days；Fig. 5 C).In general, these are protected/deposit It lives studies have shown that PD-L2 expression is preferably to control non-lethality malaria and institute of surviving from 17XNL plants of malaria of Plasmodium yoelii It is required.

Embodiment 4

PD-L2 improves the helminth specific C D4 in mouse⁺T cell response

Next the present inventor pays close attention to understanding, why when PD-L2 is blocked mouse cannot be from non-lethality Yue Shi malaria It survives in the infection that 17XNL plants of protozoon.Therefore, they repeat above-mentioned blocking experiment and the 7th day and the 14th day collection spleen with It is assessed by panimmunity measurement.Firstly, detection CD4⁺T cell T_betA kind of (Th1CD4⁺Needed for the effector function of T cell Transcription factor) expression, it is known that they can mediate protective effect (Kumar and Miller, Immunol to malaria Lett, 25:109-114,1990；Stephens and Langhorne, PLoS Pathog, 6:e1001208,2010；And Su and Stevenson, J Immunol, 168:1348-1355,2002).The CD62L for also assessing T cell (has found in T cells Marker) expression, also distinguished maincenter memory (CD62L^hi) and effect memory (CD62L^lo) T cell (Fig. 7 A).With it is initial small Mouse (the 0th day；Fig. 8 A) it compares, in the control mice for giving rat IgG, rather than (Fig. 8 B in the mouse of PD-L2 blocking；p> 0.05), T is expressed in each spleen within the 7th day_betCD62L^hi CD4⁺The number of T cell dramatically increases (Fig. 8 B；p<0.0095). By the 14th day, the expression T that the every spleen of control mice has high 2.2 times and 3 times than giving the mouse of anti-PD-L2 antibody respectively_bet CD62L^hiAnd CD62L^loCD4+T cell (Fig. 8 C).Similarly, by parasite antigen MSP1 in culture₁₉Response It measures, the helminth specific C D4 of secretion of gamma-IFN in the 14th day control mice⁺The mouse that T cell number ratio PD-L2 is blocked is high > 5 times (Fig. 8 D).External EdU intake measurement confirms that control mice has the helminth specific C D4 of higher number⁺T cell, (Fig. 8 E) is proliferated in the response to parasite antigen.However, PD-L2 blocks the level (figure for not significantly affecting serum I FN- γ 8F).On the contrary, the mouse that PD-L2 is blocked had more twice of serum IL -10 (Fig. 8 G) than control mice by the 14th day.With compare The mouse for the treatment of is compared, the result and regulatory T cells (T in each spleen observed by PD-L2 blocking_reg) number Dramatically increase related (Fig. 8 H).

It also found in the research that the PD-L2ko mouse infected with 17XNL plants of Plasmodium yoelii carries out, it is small with the WT of infection Mouse is compared, and shows the expression T of significant lesser number in the 14th day each spleen_betAnd the helminth specific C D4 of secretion of gamma-IFN⁺T cell (Fig. 7 B and C).Finally, compared with the wt mouse of infection, infection PD-L2ko mouse or give anti-PD-L2 and block In the infecting mouse of property antibody, the helminth specific C D8 of secretion of gamma-IFN in the 14th day each spleen⁺T cell is not shown It writes and reduces (Fig. 7 D).

In short, data show PD-L2 expression for effective Th1CD4 of anti-Plasmodium yoelii 17XNL plants of malaria⁺T cell is answered It is required for answering.It is related with lower parasitemia in view of PD-L2 on DC and the higher rate that PD-L1 is expressed, and PD-L2 Blocking lead to Th1 response reduction, it is therefore assumed that PD-L2 may inhibit PD-L1 function, PD-L1, which is reported, thinks that Th1 is inhibited to answer Answer (Liang et al., Eur.J.Immunol.36:58-64,2006).In addition, PD-L2 blocking causes to infect Plasmodium yoelii 17XNL plants rather than the dead mouse of Xia Shi plasmodium malaria.In comparison, compared to Xia Shi plasmodium malaria (Horne- Debets et al. Cell.Reports.5:1204-1213,2013), it is previously displayed in 17XNL plants of Plasmodium yoelii of acute stage The immunosupress that (Butler et al., Nat.Immunol.13:188-195,2012) PD-L1/PD-1 is mediated is stronger.Therefore, I Draw a conclusion, PD-L2 mediate to PD-L1/PD-1 mediate immunosuppressive inhibition, can explain two kinds infection between The Different Results that PD-L2 is blocked.

Embodiment 5

Coexpression of the PD-L1 and PD-L2 on DC is set for using really to immune

The present inventor determines whether the expression of the PD-L1 on DC leads to the lethal of malaria followed by DC transfer research Property.For this purpose, wt and PD-L1ko mouse YM plants of malaria infections of lethal Plasmodium yoelii, the 7th day separation DC (schemes after infection 9A and eB) and it is transferred to initial mouse, then infected with YM plants of malaria of lethal Plasmodium yoelii.Due to clinical score >= 100% mouse for giving the DC from wt mouse must be euthanized in 4,10 days, all to give from PD-L1ko mouse DC mouse survival (Fig. 9 C) and remove infection (Fig. 9 D and 9E).This transfer research shows that the PD-L1 on DC causes extremely It dies, because mouse is given with abundant PD-L1 but the seldom DC (referring to Fig. 3 A, C and Fig. 4 D) of PD-L2, and cannot survive, And all mouse survivals for giving PD-L1ko DC.

Then with YM plants of infection WT and PD-1ko mouse of lethal Plasmodium yoelii to confirm that PD-1 approach is that Yue Shi malaria is former The reason of YM plants of malaria lethals of worm.Since clinical score >=4, the 10 days must be euthanized to 100% WT, own PD-1ko mouse survives (Fig. 9 F) and removes infection (Fig. 9 G and 9H), it was demonstrated that PD-1/PD-L1 approach drives Plasmodium yoelii YM The lethal of strain infection.In general, these are research shows that PD-1 and PD-L1 have mediated the lethal of malaria.

In view of PD-L2 express with from malaria survival it is related, next inventor has studied PD-L2 and PD-L1 on DC and is total to It is immune how expression is adjusted.Before studies have shown that mutual between PD-1 in PD-L1 and CD8+OTI T cell on DC Effect cause ligand induce T cell receptor (TCR) lower (Karwacz et al., EMBO.Mol.Med.3:581-592, 2011).It is tested to study to reconcile under the TCR that whether PD-L2 and PD-L1 coexpression can inhibit PD-L1 to mediate on DC and lure Conductivity type T cell costimulation object (ICOS) expression.For this purpose, the DC and T cell of mouse (1:5 cell) culture purified from infection, are used Antibody blocking PD-1, PD-L1 or PD-L2 function, and CD3 high expression, TCR component and the height of T cell are detected after 36 hours ICOS expression, can indicate T cell activation (Fig. 4 I-4N).In DC:T cell culture, with anti-PD-1 antibody blocking PD-1 Signal transduction dramatically increases the expression of CD3 and ICOS, shows that PD-1 signal lowers table of these molecules in T cell to T cell Up to (Fig. 4 K and 4N).When with antibody blocking PD-L1 signal, when only PD-L2 being made to work, T cell has the ICOS dramatically increased It is horizontal (Fig. 4 L and 4N) with CD3.On the contrary, when keeping PD-L1 fully functional, CD3 and ICOS significantly lose (Fig. 4 M when blocking PD-L2 And 4N).In short, these discoveries show in the cell from 17XNL plants of infecting mouses of Plasmodium yoelii, the PD-L1 table on DC Up to may inhibit T cell activation, and PD-L2 seems to promote CD3 and ICOS expression.

Material and method

DC transfer research

CD11c⁺DC is obtained from 10⁴The spleen of the wt and PD-L1ko mouse of YM plants of (lethal) pRBC of Plasmodium yoelii infection. 4 days after infection, mouse is treated with 250 μ g pyrimethanils daily, continues 4 days to remove infection.It is rich using Dynal DC at the 7th day Collection kit digestion spleen is simultaneously enriched with DC.Run sample is on AutoMAC to remove remaining Dynal label cell and plasmodium Pigment.By marking DC with anti-CD11c MACS pearl and separating on AutoMACS, highly purified DC is obtained.It then will about 1.5×10⁷DC intravenous infusion is to initial mouse.It is former with the Yue Shi malaria of lethal dose after mouse tranquillization 15 hours or more YM plants (10 of worm⁴PRBC) infect them.When monitoring stopping, tracking mouse 48 days.

DC-T cell culture

With 10⁵17XNL plants of pRBC infecting mouses of Plasmodium yoelii, and the 14th day after infection, it digests spleen and uses CD90.2MACS pearl separates total T cell, to minimize any influence to TCR.Then using Dynal DC enrichment kit from DC is separated in remaining splenocyte.

In at least triplicate hole, with about 2 × 10⁵A DC culture about 10⁶A T cell.Control or the anti-PD-1 of blocking property (RMP1-14), anti-PD-L1 (10F.9G2) or anti-PD-L2 (TY25) antibody are added in culture with the concentration of 20 μ g/ml.Training After supporting 36 hours, cell is washed, and be marked for flow cytometry.In CD4 living⁺CD62L^loPD-1⁺It is assessed in T cell CD3 and ICOS expression.

Embodiment 6

The expression of PD-L2 on the DC of metastatic cancer patient

In order to provide additional Proof of Concept data, compare from the patient with benign lesion or metastasis melanin tumor Blood DC.PD-L2 is observed in the patient with metastatic disease⁺The significant forfeiture of DC (referring to Figure 10).Although healthy The ratio of volunteer %PD-L2:%PD-L1 is about 0.9, but the ratio drops to 0.4 to 0.8 during metastasis melanin tumor Between.It is interesting that %PD-L2:%PD-L1 ratio increases to 0.9 in the patient with local patholoic change (i.e. benign tumour) To between 1.3.

Discovery based on systemic malaria and cancer, it appears that PD-L2, but be not PD-L1 prediction Th1 immune response phase relation System property the severity of disease.In general, prediction PD-L2 level increases during autoimmune disease, and driving is destructive The amplification of immune effector cell.The patient of local patholoic change with higher proportion reflects this point.

Embodiment 7

PD-L2 multimerization indicates TH1 immune state

The present inventor assumes that multimerization solubility PD-L2 (sPD-L2) will compete PD-L1 to combine the PD- on Th1 cell 1, to reduce PD-L1 to the inhibiting effect of T cell function.In order to test this point, generates to contain and melt with the part human IgG Fc The Plasmid Constructs of the mouse PD-L2 extracellular domain of conjunction, and pass through Geneart (Life Technologies；Germany) turn It contaminates in mammalian cell.Using Protein G column from culture supernatants purification of soluble dimerization PD-L2Ig albumen (PD- L2).The albumen, which is shown, to be had < 0.2EU/mL endotoxin.According to the explanation of manufacturer, by with EZ- connection-sulfo group-NHS The biotinylation dimer PD-L2 of biotin makes PD-L2 multimerization, as measured by kit, obtains every PD-L2 dimer 2-5 biotin molecule, it is horizontal (Pierce, US) to measure biotinylation.By keeping albumen excessive by the removing of PD-10 column Biotin.Biotinylated PD-L2 and Streptavidin (Cedarlane, US) are mixed with 4:1 molar ratio, generate multimerization PD-L2 chimeric polyeptides, wherein most are eight dimer forms.Every batch of Streptavidin is tested by albuminometry, because small The amount provided in bottle is more than purchase volume.Ratio optimization is carried out for every batch of Streptavidin, because every batch of has different work Property (for example, 6:1).The western blot research carried out with the natural PAGE gel of low percentage confirms that protein multimerisation is tool Eight aggressiveness of about 300-400kDa band.

Next, with YM plants of lethal Plasmodium yoelii or Bai Shi mouse Infected With Plasmodium wild-type mice, and in helminth Mass formed by blood stasis can measure after the 3rd day, then the 5th day and the 7th day application PD-L2 after infection.It is used in combination with YM plants of infection of Plasmodium yoelii All wild-type mices of control human IgG (control Ig) treatment are dead or (Figure 11 A-C) must be euthanized in 10 days.

Similarly, dimerization PD-L2 does not provide any protective effect (Figure 11 D) to increased parasitemia.On the contrary, It is survived with the mouse (n=12) of 92% YM plants of Plasmodium yoelii infection of PD-L2 treatment, and with less symptom in 25 days Remove infection (Figure 11 A-E).By all survival mice tranquillization to the 150th day, together with the initial control mouse of new age-matched (control Ig-R) together, is attacked again with YM plants of malaria of lethal Plasmodium yoelii of same dose and (is not applied additional PD- L2；Figure 11 A).It was previously survived from infecting again in the case where no symptom with all mouse that PD-L2 is treated, and 8 small There was only 4 any parasitemias of display in mouse, as shown in the logarithmic scale in Figure 11 B.After infecting again in 20 days, 80% These fully erased infection of infecting mouses again through sPD-L2 treatment, because shifting 200 μ l from these mouse to initial mouse Blood does not shift infection (Figure 11 F).In contrast, the control mice of second group of age-matched dies of infection, it was confirmed that is used for The lethal (Figure 116 C) of the helminth infected again.In short, multimerization PD-L2 can be with after with YM plants of Plasmodium yoelii infection The lethality for overcoming PD-L1 to mediate.

Similarly, the control mice of 100% infection Bai Shi mouse plasmodium develops the symptom of experimental cerebral malaria in 8 days (ECM) (Figure 11 G), and infection (Figure 11 H) was died of at the 10th day.With the mouse of the Bai Shi mouse Infected With Plasmodium of sPD-L2 treatment There is 22% generation cerebral malaria, as seen in ECM scoring (Figure 11 G).In addition, survival mice control about 20 days (Figure 11 H and I) of infection, 13 days after dying of last one PD-L2.Additional dosage does not improve survival (data are not shown).In short, application multimerization PD-L2 significantly improves the survival from lethal infection, and reduces the severity of clinical symptoms, especially for brain malaria Disease.

Embodiment 8

Ten dimerization PD-L2 enhance the immune response to tumour

Soluble chimeric PD-L2 polypeptide is designed, ten dimers are self-assembled into.The chimeric polyeptides contain and the portion human IgG Fc Divide the mouse PD-L2 extracellular domain and the end C- α-tail portion (mouse PD-L2-Fc-atp) of fusion, and there is following ammonia Base acid sequence:

The sequence of capitalization and underline font styles represents the amino acid sequence of mouse PD-L2 signal peptide；The sequence of small letters Amino acid sequence corresponding to mouse PD-L2 extracellular domain；The sequence of italic small letters represents mouse PD-L2 transmembrane domain portion The amino acid sequence divided；The sequence of uppercase corresponds to the amino acid sequence of the Fc polypeptide of human IgG1；And bold upper case letters The sequence of body represents the amino acid sequence of IgA molecule alpha tail portion (α tp).

The construct of the DNA sequence dna of encoding chimera polypeptide is expressed by Geneart (Life Technologies；Germany) turn It contaminates in exclusive mammalian cell, and purifies ten dimerization PD- of most of solubility from culture supernatant using Protein G column L2-Ig albumen (sPD-L2), then FPLC is fractionated to exclude the dimeric forms of albumen.The albumen, which is shown, to be had < 0.2EU/mL Endotoxin.

Next, B16.F0 the and B16.F10 K-1735 established is grown in the lab, and in C57BL/ It is subcutaneous in 6 mouse to be implanted into 5 × 10 respectively⁵Or 1 × 10⁵A cell.

In order to determine effect of the sPD-L2 to advanced melanoma, when B16.F0 tumour reached 100mm at the about the 9th day³Body When product, 200 μ g human IgG of mouse or sPD-L2, and every 1-2 days monitoring tumor sizes were given at the 9th, 11 and 13 day.For human relations Reason is managed, if tumour > 1000mm³Or showing any ulcer or malaise symptoms, then mouse is euthanized.The result of the research is such as Shown in Figure 12, clearly illustrate that sPD-L2 enhancing generates guarantor to the immune response of advanced melanoma, and to advanced melanoma Shield effect.

It is investigated effect of the sPD-L2 to infantile tumour.In these experiments, tumour reaches about 50mm³When volume, About the 3rd day or so implantation B16.F10 cell, and mouse was treated with 200 μ g human IgGs or sPD-L2 at the 3rd, 9 and 15 day.This grinds The result (referring to Figure 13) studied carefully shows that sPD-L2 provides the early protection for being directed to melanoma.

The disclosure of herein cited every patent, patent application and publication is incorporated herein by reference in their entirety.

The reference of any bibliography is not necessarily to be construed as recognizing such with reference to " prior art " that can be used as the application.

Throughout the specification, it is therefore an objective to describe the preferred embodiments of the invention, rather than limit the invention to appoint What embodiment or specific characteristic set.Therefore, it will be understood by those skilled in the art that according to present disclosure, not In the case where being detached from the scope of the invention, exemplary specific embodiment can be carry out various modifications and be changed.It is all these to repair Change and change is included in scope of the appended claims.

Claims (56)

1. a kind of polypeptide complex is shown in formula (I):

[P]_n (I)

Wherein:

P, independently represents protein molecular when occurring every time, the protein molecular includes PD-L2 polypeptide, be made of PD-L2 polypeptide, Or it is substantially made of PD-L2 polypeptide；And

N represents the integer greater than 2.

2. polypeptide complex according to claim 1, wherein P lacks tumour or the relevant new vessels targeting structure of tumour Domain.

3. according to claim 1 or polypeptide complex as claimed in claim 2, wherein the PD-L2 polypeptide include PD-L2 can Molten part is made of the soluble fraction of PD-L2 or is substantially made of the soluble fraction of PD-L2.

4. polypeptide complex according to claim 3, wherein the soluble fraction of the PD-L2 is the PD-L2 for having signal peptide The PD-L2 extracellular domain of extracellular domain or not signal peptide.

5. polypeptide complex according to any one of claim 1 to 4, wherein the protein molecular lacks PD-L2 cross-film One or both of structural domain and PD-L2 cytoplasmic domains.

6. polypeptide complex according to any one of claim 1 to 5, wherein n be >=3, >=4, >=5, >=6, >=7 or >= 8。

7. polypeptide complex according to any one of claim 1 to 6, wherein n be≤100 ,≤50 ,≤30 or≤20.

8. polypeptide complex according to any one of claim 1 to 7, it is suitably 4 to 16, more that wherein n, which is 3 to 20, It is suitably 8 to 12.

9. polypeptide complex according to any one of claim 1 to 8, wherein the protein molecular chemical coupling is together To form the polypeptide complex.

10. polypeptide complex according to any one of claim 1 to 8, wherein single protein molecular also includes at least one A oligomerization domain, the oligomerization domain promote the self assembly of the protein molecular to form the polypeptide complex.

11. polypeptide complex according to claim 10, wherein at least one described oligomerization domain and the PD-L2 Polypeptide is operably connected to form single-chain chimeric polypeptide.

12. a kind of protein molecular, it includes PD-L2 polypeptide defined by any one of claims 1 to 9, by claim 1 to PD-L2 polypeptide defined by any one of 9 forms or substantially the PD-L2 as defined by any one of claims 1 to 9 is more Peptide composition, it is compound to form polypeptide shown in formula (I) that the PD-L2 polypeptide is operably coupled to a few oligomerization domain Object, wherein the oligomerization domain promotes the self assembly of the protein molecular.

13. polypeptide complex according to any one of claims 10 to 12, wherein at least one described oligomerization structure Domain is operably coupled to upstream (i.e. amino terminal) and/or downstream (i.e. the carboxyl terminal) of the PD-L2 polypeptide.

14. polypeptide complex according to claim 13, wherein the protein molecular includes single polypeptide shown in formula (II) Chain, the single polypeptide chain shown in formula (II) form or substantially the single polypeptide chain shown in formula (II) forms:

PD-L²-L-OMD_A (II)

Wherein:

PD-L2 represents PD-L2 polypeptide；

OMD_AIt is oligomerization domain, forms i subunit OMD_AOligomer (OMD_A)_i, wherein i >=3 are suitably 3,4,5 Or 6；And

L is key or peptide linker.

15. polypeptide complex according to claim 13, wherein the protein molecular includes single polypeptide shown in formula (III) Chain, the single polypeptide chain shown in formula (III) form or substantially the single polypeptide chain shown in formula (III) forms:

PD-L2-L-OMD_A-L-OMD_B (III)

Wherein:

OMD_AIt is oligomerization domain, forms i subunit OMD_AOligomer (OMD_A)_i, wherein i >=2 are suitably 2,3,4, 5 or 6；

L independently represents key or peptide linker when occurring every time；And

OMD_BIt is oligomerization domain, forms j subunit OMD_BOligomer (OMD_B)_j, wherein j is greater than the integer of i, suitably Ground is i+1, i+2, i+3, i+4, i+5 or i+6.

16. polypeptide complex according to claim 15, wherein i is 2 and j is 4 or 6.

17. polypeptide complex according to claim 13, wherein the protein molecular includes single polypeptide shown in formula (IV) Chain, the single polypeptide chain shown in formula (IV) form or substantially the single polypeptide chain shown in formula (IV) forms:

OMD_A-L-PD-L2 (IV)

Wherein:

OMD_AIt is oligomerization domain, forms i subunit OMD_AOligomer (OMD_A)_i, wherein i >=3 are suitably 3,4,5 Or 6；

L is key or peptide linker；And

PD-L2 represents PD-L2 polypeptide.

18. polypeptide complex according to claim 13, wherein the protein molecular includes single polypeptide shown in formula (V) Chain, the single polypeptide chain shown in formula (V) form or substantially the single polypeptide chain shown in formula (V) forms:

OMD_B-L-OMD_A-L-PD-L2 (V)

Wherein:

OMD_BIt is oligomerization domain, forms j subunit OMD_BOligomer (OMD_B)_j, wherein j >=2 are suitably 2,3,4, 5 or 6；

L independently represents key or peptide linker when occurring every time；And

OMD_AIt is oligomerization domain, forms i subunit OMD_AOligomer (OMD_A)_i, wherein i is greater than the integer of j, suitably Ground is j+1, j+2, j+3, j+4, j+5 or j+6；And

PD-L2 represents PD-L2 polypeptide.

19. polypeptide complex according to claim 18, wherein j is 2 and i is 4 or 6.

20. polypeptide complex described in any one of 4 to 19 according to claim 1, wherein single in the presence of binding partners Oligomerization domain is (for example, OMD_AOr OMD_B) it is assembled into different oligomer.

21. polypeptide complex according to claim 20, wherein the oligomerization domain and the binding partners can To be the member of specific binding pair.

22. polypeptide complex according to claim 21, wherein the specific binding is to being selected from: biotin-antibiont Fibroin, biotin-Streptavidin, Ag-Ab, haptens-antihapten, ligand-receptor and receptor-co-receptor.

23. polypeptide complex described in any one of 0 to 22 according to claim 1, wherein at least one described oligomerization structure Domain is selected from: dimerization domain (for example, immunoglobulin Fc domain, leucine zipper etc.), trimerising domain (for example, The catalytic subunit of Escherichia coli aspartate carbamyl-transferase (ATCase), from T4 bacteriophage minority fibers albumen " foldon " trimerization sequence, neck region peptide, people's Curosurf protein D, oligomerization coiled coil adhesin, I class enveloped virus Complementary heptad repeat region of fusion protein etc.), tetramerization structural domain (for example, coiled-coil domain of the viscous connection albumen of four arms), Five multimerisation domains (for example, tryptophan zipper or five multimerisation domains of cartilage oligo-substrate protein (COMP) etc.) and six dimerizations Structural domain (for example, tail portion from IgA antibody heavy chain C-terminal).

24. polypeptide complex described in any one of 0 to 23 according to claim 1, wherein at least one described oligomerization structure Domain is directly connected to the PD-L2 polypeptide.

25. polypeptide complex described in any one of 0 to 23 according to claim 1, wherein at least one described oligomerization structure Domain is connected with the PD-L2 polypeptide by peptide linker.

26. polypeptide complex according to claim 25, wherein the peptide linker is residual by about 1 to about 100 amino acid Base (and all integer amino acid residues therebetween) composition.

27. according to polypeptide complex described in claim 25 or claim 26, wherein the peptide linker includes at least one Part, the part are selected from: being promoted to purify the purification part of the protein molecular, be adjusted to the protein molecular immune response Immunological regulation part and the part for assigning configuration flexibility.

28. a kind of nucleic acid construct, it includes coded sequence, any one of described coded sequence coding claim 10 to 27 institute The protein molecular of restriction, the coded sequence are operably coupled to regulating element, and the regulating element is in host cell It is operable.

29. a kind of host cell contains the nucleic acid construct described in claim 28.

30. host according to claim 29 is prokaryotic host cell.

31. host according to claim 29 is eukaryotic host cell.

32. a kind of method for generating polypeptide complex, which comprises (example under conditions of suitably forming polypeptide complex Such as, in aqueous solution), protein molecular defined by any one of claim 10 to 27 is combined, to generate polypeptide Compound, it includes the oligomer of n protein subunit molecule, wherein n usually >=3, >=4, >=5, >=6, >=7 or >=8, suitably Wherein n≤100 ,≤50 ,≤30 or≤20, n is 3 to 20 preferably wherein, is suitably 4 to 16, is more suitably 8 to 12.

33. a kind of immune regulation composite, it includes polypeptide complexes defined by any one of claim 1 to 27, and Pharmaceutically acceptable carrier, diluent or adjuvant.

34. a kind of in subject's moderate stimulation, initiation or the method for enhancing immune response, the immune response, which includes that Th1 is immune, is answered It answers, the method comprise the steps that it is compound to apply polypeptide defined by any one of claim 1 to 27 or 33 to the subject Object or composition.

35. a kind of treat the method for Th1 related disease or illness in subject, which comprises Xiang Suoshu subject's application Polypeptide complex defined by any one of a effective amount of claim 1 to 27 or 33 or composition.

36. according to method described in claim 34 or claim 35, wherein being damaged when the subject is accredited as having Th1 it is immune when, the polypeptide complex or composition are applied to subject.

37. according to the method for claim 36, further includes: assess the Th1 immune state of subject by the following method: (1) the Th1 immune state biomarker spectrum being obtained from the sample of subject is determined, wherein the Th1 immune state biology mark Remember biomarker value of the object spectrum comprising at least one of sample Th1 immune state biomarker, wherein it is described at least A kind of Th1 immune state biomarker includes and immune effector cell (IEC)-interacts cell phase interaction in the sample PD-L2 and optional PD-L1 in cell；(2) indicant is determined using the biomarker value, wherein described Indicant at least partly indicates the Th1 immune state of the subject.

38. according to the method for claim 37, wherein IEC- interaction cell is antigen presenting cell (APC), It is appropriately selected from dendritic cells and macrophage.

39. according to the method for claim 38, wherein the APC is the dendritic cells for expressing CD11c.

40. according to the method for claim 37, wherein IEC- interaction cell is tumour cell.

41. the method according to any one of claim 37 to 40, wherein the biomarker value at least partly refers to Show the concentration of Th1 immune state biomarker in the sample obtained from subject.

42. according to the method for claim 41, wherein the biomarker value includes the Th1 immune state biology mark Remember the abundance of object.

43. the method according to any one of claim 37 to 42, wherein individual biomarker value includes: in cell table The percentage of the antigen presenting cell of Th1 immune state biomarker is expressed on face (for example, PD-L2⁺Dendritic cells, PD-L2⁺ Tumour cell etc.).

44. according to the method for claim 40, wherein the Th1 immune state biomarker is PD-L2, and described Biomarker value is collected on the PD- on IEC- interaction surface cell (for example, APC such as dendritic cells, tumour cell) The measurement of L2.

45. the method according to any one of claim 37 to 44, wherein for the control level of PD-L2, sample The horizontal of PD-L2 reduces in product, wherein the control level of the PD-L2 be immunized there are normal Th1 or undamaged Th1 exempts from Epidemic disease is related, and determines that the indicant at least partly indicates Th1 immunocompromised host.

46. the method according to any one of claim 37 to 45, wherein coming from a pair of Th1 immune state biomarker Biomarker value for determining indicant.

47. method described in claim 46, wherein the pair of biomarker is PD-L2 and PD-L1.

48. according to the method for claim 47, further including that combination function is applied to the biomarker value.

49. according to the method for claim 48, wherein the combination function is selected from: additive model, linear model, support to Amount machine, neural network model, Random Forest model, regression model, genetic algorithm, annealing algorithm, weighted sum, Neighborhood Model and general Rate model.

50. the method according to any one of claim 37 to 46, comprising: (a) determines the first Th1 immune state biology mark Remember the biomarker value of object；(b) the corresponding biomarker value of the 2nd Th1 immune state biomarker is determined；(c) it uses The biomarker value of first and second Th1 immune state biomarkers record determines indicant, the indicant instruction The first and second Th1 immune state biomarkers record biomarker value ratio.

51. according to the method for claim 50, wherein the first Th1 immune state biomarker is PD-L2, and The 2nd Th1 immune state biomarker is PD-L1.

52. according to method described in claim 50 or claim 51, wherein relative to control PD-L2:PD-L1 biology mark For the ratio for remembering object value, ratio (" the sample Th1 of the first and second Th1 immune state biomarker values determined in sample The ratio of immune state biomarker ") reduce, wherein it is described control PD-L2:PD-L1 biomarker value ratio with deposit In the immune correlation of normal Th1 immune or undamaged Th1, and determine the indicant at least partly indicate Th1 be immunized by Damage.

53. method according to claim 52, wherein suitably, the ratio of the biomarker value of sample is no more than pair According to biomarker value ratio (for example, from tested with normal Th1 immune response or undamaged Th1 immune response In the resulting control sample of person determine) about 95%, 94%, 93%, 92%, 91%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% (and each integer therebetween).

54. method according to claim 53, wherein the ratio of the PD-L2:PD-L1 biomarker value of the sample is logical It is often the ratio of control biomarker value (for example, from normal Th1 immune response or undamaged Th1 immune response The resulting control sample of subject in determine) about 96% to 104% (and all integer percents therebetween).

55. the method according to any one of claim 35 to 54, wherein the Th1 related disease or illness and reduction Th1 immune state or the Th1 immune state of inhibition are related.

56. method according to claim 55, wherein the Th1 related disease or illness are cancer or pathogenic infection, The cancer includes metastatic cancer.